Virulence, antimicrobial susceptibility and phylogenetic analysis of Cronobacter sakazakii isolates of food origins from Jordan.

AIMS: The aim was to characterize a collection of Cronobacter sakazakii isolates collected from various origins in Jordan. METHODS AND RESULTS: The isolates were characterized using 16S rRNA sequencing, DNA microarray, multi-locus sequence typing (MLST), O-serotyping, virulence gene identification and antibiotic susceptibility testing. The identities and phylogenetic relatedness revealed that C. sakazakii sequence type 4 (ST4) and Csak O:1 serotype were the most prevalent STs and serovars amongst these C. sakazakii strains. PCR screening of putative virulence genes showed that the siderophore-interacting protein gene (sip) and iron acquisition gene clusters (eitCBAD and iucABCD/iutA) were the most detected genes with noticeable variability in the type 6 secretion system (T6SS) and filamentous hemagglutinin/adhesion (FHA) gene loci. The antibiotic resistance profiles revealed that the majority of the isolates were susceptible to all antibiotics used despite harbouring a class C ß-lactamase resistance gene. CONCLUSIONS: The results described in this report provide additional insights about the considerable genotypic and phenotypic heterogeneity within C. sakazakii. SIGNIFICANCE AND IMPACT OF THE STUDY: The information reported in this study might be of great value in understanding the origins of C. sakazakii isolates, in addition to their diversity and variability, which might be helpful in preventing future outbreaks of this pathogen.

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.

Culture-Based Quantification with Molecular Characterization of Non-O157 and O157 Enterohemorrhagic Escherichia coli Isolates from Rectoanal Mucosal Swabs of Feedlot Cattle.

Enterohemorrhagic Escherichia coli (EHEC) strains are foodborne pathogens carried in the intestinal tracts of ruminants and shed in the feces. High concentrations (≥104 colony-forming units [CFU]/g) of EHEC in cattle feces are associated with contamination of hides, and subsequently, carcasses and beef. Several studies using agar media have quantified O157 but few have quantified non-O157 EHEC in samples from cattle. Thus, the objective of this study was to determine the concentration of O157 and non-O157 EHEC in cattle, and to characterize the associated EHEC isolates for their virulence potential. Two hundred feedlot steers were sampled by rectoanal mucosal swab (RAMS) every 35 days over four sampling periods, and a spiral plating method using modified Possé differential agar was used to quantify EHEC organisms in these samples. Bacterial colonies from agar plates were tested by multiplex PCR for Shiga toxin and intimin genes (stx and eae, respectively), and confirmed EHEC isolates (i.e., positive for both stx and eae) were serotyped and characterized for virulence genes using a microarray. Organisms detected in this study included O26, O101, O103, O109, O121, O145, O157, and O177 EHEC, with all except O121 quantifiable and measuring within a range from 9.0 × 102 to 3.0 × 105 CFU/g of RAMS sample. Organisms of the same EHEC serogroup were not detected in quantifiable concentrations from a single animal more than once. EHEC organisms most commonly detected at quantifiable levels were O26, O157, and O177. Interestingly, O26 EHEC isolates tested negative for stx1 but positive for stx2a. High concentrations of EHEC were detected in 11 (5.5%) of the steers at least once over the sampling period. These results indicate that in addition to O157, non-O157 EHEC are transiently present in high concentrations in the rectoanal mucosal region of cattle.

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.

Variability and bias in microbiome metagenomic sequencing: an interlaboratory study comparing experimental protocols.

Several studies have documented the significant impact of methodological choices in microbiome analyses. The myriad of methodological options available complicate the replication of results and generally limit the comparability of findings between independent studies that use differing techniques and measurement pipelines. Here we describe the Mosaic Standards Challenge (MSC), an international interlaboratory study designed to assess the impact of methodological variables on the results. The MSC did not prescribe methods but rather asked participating labs to analyze 7 shared reference samples (5 × human stool samples and 2 × mock communities) using their standard laboratory methods. To capture the array of methodological variables, each participating lab completed a metadata reporting sheet that included 100 different questions regarding the details of their protocol. The goal of this study was to survey the methodological landscape for microbiome metagenomic sequencing (MGS) analyses and the impact of methodological decisions on metagenomic sequencing results. A total of 44 labs participated in the MSC by submitting results (16S or WGS) along with accompanying metadata; thirty 16S rRNA gene amplicon datasets and 14 WGS datasets were collected. The inclusion of two types of reference materials (human stool and mock communities) enabled analysis of both MGS measurement variability between different protocols using the biologically-relevant stool samples, and MGS bias with respect to ground truth values using the DNA mixtures. Owing to the compositional nature of MGS measurements, analyses were conducted on the ratio of Firmicutes: Bacteroidetes allowing us to directly apply common statistical methods. The resulting analysis demonstrated that protocol choices have significant effects, including both bias of the MGS measurement associated with a particular methodological choices, as well as effects on measurement robustness as observed through the spread of results between labs making similar methodological choices. In the analysis of the DNA mock communities, MGS measurement bias was observed even when there was general consensus among the participating laboratories. This study was the result of a collaborative effort that included academic, commercial, and government labs. In addition to highlighting the impact of different methodological decisions on MGS result comparability, this work also provides insights for consideration in future microbiome measurement study design.

Genetic analysis of the roles of agaA, agaI, and agaS genes in the N-acetyl-D-galactosamine and D-galactosamine catabolic pathways in Escherichia coli strains O157:H7 and C.

BACKGROUND: The catabolic pathways of N-acetyl-D-galactosamine (Aga) and D-galactosamine (Gam) in E. coli were proposed from bioinformatic analysis of the aga/gam regulon in E. coli K-12 and later from studies using E. coli C. Of the thirteen genes in this cluster, the roles of agaA, agaI, and agaS predicted to code for Aga-6-P-deacetylase, Gam-6-P deaminase/isomerase, and ketose-aldolase isomerase, respectively, have not been experimentally tested. Here we study their roles in Aga and Gam utilization in E. coli O157:H7 and in E. coli C. RESULTS: Knockout mutants in agaA, agaI, and agaS were constructed to test their roles in Aga and Gam utilization. Knockout mutants in the N-acetylglucosamine (GlcNAc) pathway genes nagA and nagB coding for GlcNAc-6-P deacetylase and glucosamine-6-P deaminase/isomerase, respectively, and double knockout mutants ΔagaA ΔnagA and ∆agaI ∆nagB were also constructed to investigate if there is any interplay of these enzymes between the Aga/Gam and the GlcNAc pathways. It is shown that Aga utilization was unaffected in ΔagaA mutants but ΔagaA ΔnagA mutants were blocked in Aga and GlcNAc utilization. E. coli C ΔnagA could not grow on GlcNAc but could grow when the aga/gam regulon was constitutively expressed. Complementation of ΔagaA ΔnagA mutants with either agaA or nagA resulted in growth on both Aga and GlcNAc. It was also found that ΔagaI, ΔnagB, and ∆agaI ΔnagB mutants were unaffected in utilization of Aga and Gam. Importantly, ΔagaS mutants were blocked in Aga and Gam utilization. Expression analysis of relevant genes in these strains with different genetic backgrounds by real time RT-PCR supported these observations. CONCLUSIONS: Aga utilization was not affected in ΔagaA mutants because nagA was expressed and substituted for agaA. Complementation of ΔagaA ΔnagA mutants with either agaA or nagA also showed that both agaA and nagA can substitute for each other. The ∆agaI, ∆nagB, and ∆agaI ∆nagB mutants were not affected in Aga and Gam utilization indicating that neither agaI nor nagB is involved in the deamination and isomerization of Gam-6-P. We propose that agaS codes for Gam-6-P deaminase/isomerase in the Aga/Gam pathway.

Rapid genomic-scale analysis of Escherichia coli O104:H4 by using high-resolution alternative methods to next-generation sequencing.

Two technologies, involving DNA microarray and optical mapping, were used to quickly assess gene content and genomic architecture of recent emergent Escherichia coli O104:H4 and related strains. In real-time outbreak investigations, these technologies can provide congruent perspectives on strain, serotype, and pathotype relationships. Our data demonstrated clear discrimination between clinically, temporally, and geographically distinct O104:H4 isolates and rapid characterization of strain differences.

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.

Characterization of Cronobacter sakazakii Strains Originating from Plant-Origin Foods Using Comparative Genomic Analyses and Zebrafish Infectivity Studies.

Cronobacter sakazakii continues to be isolated from ready-to-eat fresh and frozen produce, flours, dairy powders, cereals, nuts, and spices, in addition to the conventional sources of powdered infant formulae (PIF) and PIF production environments. To understand the sequence diversity, phylogenetic relationship, and virulence of C. sakazakii originating from plant-origin foods, comparative molecular and genomic analyses, and zebrafish infection (ZI) studies were applied to 88 strains. Whole genome sequences of the strains were generated for detailed bioinformatic analysis. PCR analysis showed that all strains possessed a pESA3-like virulence plasmid similar to reference C. sakazakii clinical strain BAA-894. Core genome analysis confirmed a shared genomic backbone with other C. sakazakii strains from food, clinical and environmental strains. Emerging nucleotide diversity in these plant-origin strains was highlighted using single nucleotide polymorphic alleles in 2000 core genes. DNA hybridization analyses using a pan-genomic microarray showed that these strains clustered according to sequence types (STs) identified by multi-locus sequence typing (MLST). PHASTER analysis identified 185 intact prophage gene clusters encompassing 22 different prophages, including three intact Cronobacter prophages: ENT47670, ENT39118, and phiES15. AMRFinderPlus analysis identified the CSA family class C ß-lactamase gene in all strains and a plasmid-borne mcr-9.1 gene was identified in three strains. ZI studies showed that some plant-origin C. sakazakii display virulence comparable to clinical strains. Finding virulent plant-origin C. sakazakii possessing significant genomic features of clinically relevant STs suggests that these foods can serve as potential transmission vehicles and supports widening the scope of continued surveillance for this important foodborne pathogen.

Investigating the global genomic diversity of Escherichia coli using a multi-genome DNA microarray platform with novel gene prediction strategies.

BACKGROUND: The gene content of a diverse group of 183 unique Escherichia coli and Shigella isolates was determined using the Affymetrix GeneChip® E. coli Genome 2.0 Array, originally designed for transcriptome analysis, as a genotyping tool. The probe set design utilized by this array provided the opportunity to determine the gene content of each strain very accurately and reliably. This array constitutes 10,112 independent genes representing four individual E. coli genomes, therefore providing the ability to survey genes of several different pathogen types. The entire ECOR collection, 80 EHEC-like isolates, and a diverse set of isolates from our FDA strain repository were included in our analysis. RESULTS: From this study we were able to define sets of genes that correspond to, and therefore define, the EHEC pathogen type. Furthermore, our sampling of 63 unique strains of O157:H7 showed the ability of this array to discriminate between closely related strains. We found that individual strains of O157:H7 differed, on average, by 197 probe sets. Finally, we describe an analysis method that utilizes the power of the probe sets to determine accurately the presence/absence of each gene represented on this array. CONCLUSIONS: These elements provide insights into understanding the microbial diversity that exists within extant E. coli populations. Moreover, these data demonstrate that this novel microarray-based analysis is a powerful tool in the field of molecular epidemiology and the newly emerging field of microbial forensics.

An FDA bioinformatics tool for microbial genomics research on molecular characterization of bacterial foodborne pathogens using microarrays.

BACKGROUND: Advances in microbial genomics and bioinformatics are offering greater insights into the emergence and spread of foodborne pathogens in outbreak scenarios. The Food and Drug Administration (FDA) has developed a genomics tool, ArrayTrack™, which provides extensive functionalities to manage, analyze, and interpret genomic data for mammalian species. ArrayTrack™ has been widely adopted by the research community and used for pharmacogenomics data review in the FDA's Voluntary Genomics Data Submission program. RESULTS: ArrayTrack™ has been extended to manage and analyze genomics data from bacterial pathogens of human, animal, and food origin. It was populated with bioinformatics data from public databases such as NCBI, Swiss-Prot, KEGG Pathway, and Gene Ontology to facilitate pathogen detection and characterization. ArrayTrack™'s data processing and visualization tools were enhanced with analysis capabilities designed specifically for microbial genomics including flag-based hierarchical clustering analysis (HCA), flag concordance heat maps, and mixed scatter plots. These specific functionalities were evaluated on data generated from a custom Affymetrix array (FDA-ECSG) previously developed within the FDA. The FDA-ECSG array represents 32 complete genomes of Escherichia coli and Shigella. The new functions were also used to analyze microarray data focusing on antimicrobial resistance genes from Salmonella isolates in a poultry production environment using a universal antimicrobial resistance microarray developed by the United States Department of Agriculture (USDA). CONCLUSION: The application of ArrayTrack™ to different microarray platforms demonstrates its utility in microbial genomics research, and thus will improve the capabilities of the FDA to rapidly identify foodborne bacteria and their genetic traits (e.g., antimicrobial resistance, virulence, etc.) during outbreak investigations. ArrayTrack™ is free to use and available to public, private, and academic researchers at http://www.fda.gov/ArrayTrack.

The Secretion of Toxins and Other Exoproteins of Cronobacter: Role in Virulence, Adaption, and Persistence.

: Cronobacter species are considered an opportunistic group of foodborne pathogenic bacteria capable of causing both intestinal and systemic human disease. This review describes common virulence themes shared among the seven Cronobacter species and describes multiple exoproteins secreted by Cronobacter, many of which are bacterial toxins that may play a role in human disease. The review will particularly concentrate on the virulence factors secreted by C. sakazakii, C. malonaticus, and C. turicensis, which are the primary human pathogens of interest. It has been discovered that various species-specific virulence factors adversely affect a wide range of eukaryotic cell processes including protein synthesis, cell division, and ion secretion. Many of these factors are toxins which have been shown to also modulate the host immune response. These factors are encoded on a variety of mobile genetic elements such as plasmids and transposons; this genomic plasticity implies ongoing re-assortment of virulence factor genes which has complicated our efforts to categorize Cronobacter into sharply defined genomic pathotypes.

Analysis of the Molecular Diversity Among Cronobacter Species Isolated From Filth Flies Using Targeted PCR, Pan Genomic DNA Microarray, and Whole Genome Sequencing Analyses.

Cronobacter species are opportunistic pathogens capable of causing life-threatening infections in humans, with serious complications arising in neonates, infants, immuno-compromised individuals, and elderly adults. The genus is comprised of seven species: Cronobacter sakazakii, Cronobacter malonaticus, Cronobacter turicensis, Cronobacter muytjensii, Cronobacter dublinensis, Cronobacter universalis, and Cronobacter condimenti. Despite a multiplicity of genomic data for the genus, little is known about likely transmission vectors. Using DNA microarray analysis, in parallel with whole genome sequencing, and targeted PCR analyses, the total gene content of two C. malonaticus, three C. turicensis, and 14 C. sakazaki isolated from various filth flies was assessed. Phylogenetic relatedness among these and other strains obtained during surveillance and outbreak investigations were comparatively assessed. Specifically, microarray analysis (MA) demonstrated its utility to cluster strains according to species-specific and sequence type (ST) phylogenetic relatedness, and that the fly strains clustered among strains obtained from clinical, food and environmental sources from United States, Europe, and Southeast Asia. This combinatorial approach was useful in data mining for virulence factor genes, and phage genes and gene clusters. In addition, results of plasmidotyping were in agreement with the species identity for each strain as determined by species-specific PCR assays, MA, and whole genome sequencing. Microarray and BLAST analyses of Cronobacter fly sequence datasets were corroborative and showed that the presence and absence of virulence factors followed species and ST evolutionary lines even though such genes were orthologous. Additionally, zebrafish infectivity studies showed that these pathotypes were as virulent to zebrafish embryos as other clinical strains. In summary, these findings support a striking phylogeny amongst fly, clinical, and surveillance strains isolated during 2010-2015, suggesting that flies are capable vectors for transmission of virulent Cronobacter spp.; they continue to circulate among United States and European populations, environments, and that this "pattern of circulation" has continued over decades.

DNA Microarray-Based Genomic Characterization of the Pathotypes of Escherichia coli O26, O45, O103, O111, and O145 Isolated from Feces of Feedlot Cattle †.

Shiga toxin-producing Escherichia coli (STEC) serogroups O26, O45, O103, O111, O121, and O145, referred to as the top six non-O157 serogroups, are responsible for more than 70% of human non-O157 STEC infections in North America. Cattle harbor non-O157 strains in the hindgut and shed them in the feces. The objective of this study was to use the U.S. Food and Drug Administration (FDA) E. coli identification (ECID) DNA microarray to identify the serotype, assess the virulence potential of each, and determine the phylogenetic relationships among five of the six non-O157 E. coli serogroups isolated from feedlot cattle feces. Forty-four strains of STEC, enterohemorrhagic E. coli (EHEC), enteropathogenic E. coli (EPEC), or putative nonpathotype E. coli (NPEC) of cattle origin and five human clinical strains of EHEC were assayed with the FDA-ECID DNA microarray. The cattle strains harbored diverse flagellar genes. The bovine and human strains belonging to serogroups O26, O45, and O103 carried stx1 only, O111 carried both stx1 and stx2, and O145 carried either stx1 or stx2. The strains were also positive for various subtypes of intimin and other adhesins (IrgA homologue adhesin, long polar fimbriae, mannose-specific adhesin, and curli). Both human and cattle strains were positive for LEE-encoded type III secretory system genes and non-LEE-encoded effector genes. SplitsTree4, a program used to determine the phylogenetic relationship among the strains, revealed that the strains within each serogroup clustered according to their pathotype. In addition to genes encoding Shiga toxins, bovine non-O157 E. coli strains possessed other major virulence genes, including those for adhesins, type III secretory system proteins, and plasmid-borne virulence genes, similar to human clinical strains. Because virulence factors encoded by these genes are involved in the pathogenesis of various pathotypes of E. coli, the bovine non-O157 strains could cause human illness. The FDA-ECID DNA microarray assay rapidly provided a profile of the virulence genes for assessment of the virulence potential of each strain.

Prevalence, Distribution, and Phylogeny of Type Two Toxin-Antitoxin Genes Possessed by Cronobacter Species where C. sakazakii Homologs Follow Sequence Type Lineages.

Cronobacter species are a group of foodborne pathogenic bacteria that cause both intestinal and systemic human disease in individuals of all age groups. Little is known about the mechanisms that Cronobacter employ to survive and persist in foods and other environments. Toxin-antitoxin (TA) genes are thought to play a role in bacterial stress physiology, as well as in the stabilization of horizontally-acquired re-combinatorial elements such as plasmids, phage, and transposons. TA systems have been implicated in the formation of a persistence phenotype in some bacterial species including Escherichia coli and Salmonella. This project's goal was to understand the phylogenetic relatedness among TA genes present in Cronobacter. Preliminary studies showed that two typical toxin genes, fic and hipA followed species evolutionary lines. A local database of 22 TA homologs was created for Cronobacter sakazakii and a Python version 3 shell script was generated to extract TA FASTA sequences present in 234 C. sakazakii genomes previously sequenced as part of Center for Food Safety and Applied Nutrition's (CFSAN) GenomeTrakr project. BLAST analysis showed that not every C. sakazakii strain possessed all twenty-two TA loci. Interestingly, some strains contained either a toxin or an antitoxin component, but not both. Five common toxin genes: ESA_00258 (parDE toxin-antitoxin family), ESA_00804 (relBE family), ESA_01887 (relBE family), ESA_03838 (relBE family), and ESA_04273 (YhfG-Fic family) were selected for PCR analysis and the primers were designed to detect these genes. PCR analysis showed that 55 of 63 strains possessed three of these genes Sequence analysis identified homologs of the target genes and some of the strains were PCR-negative for one or more of the genes, pointing to potential nucleotide polymorphisms in those loci or that these toxin genes were absent. Phylogenetic studies using a Cronobacter pan genomic microarray showed that for the most part TAs follow species evolutionary lines except for a few toxin genes possessed by some C. malonaticus and C. universalis strains; this demonstrates that some TA orthologues share a common phylogeny. Within the C. sakazakii strains, the prevalence and distribution of these TA homologs by C. sakazakii strain BAA-894 (a powdered infant formula isolate) followed sequence-type evolutionary lineages. Understanding the phylogeny of TAs among the Cronobacter species is essential to design future studies to realize the physiological mechanisms and roles for TAs in stress adaptation and persistence of Cronobacter within food matrices and food processing environments.

Draft Genome Sequences of the Escherichia coli Reference (ECOR) Collection.

Here, we report the genomes of all 72 isolates belonging to the Escherichia coli reference (ECOR) collection. Strains in this collection were isolated from diverse hosts and geographic locations and have been used for more than 30 years to represent the phylogenetic diversity of E. coli.

Genetic Analysis of Virulence Potential of Escherichia coli O104 Serotypes Isolated From Cattle Feces Using Whole Genome Sequencing.

Escherichia coli O104:H4, a Shiga toxin-producing hybrid pathotype that was implicated in a major foodborne outbreak in Germany in 2011, has not been detected in cattle. However, serotypes of O104, other than O104:H4, have been isolated from cattle feces, with O104:H7 being the most predominant. In this study, we investigated, based on whole genome sequence analyses, the virulence potential of E. coli O104 strains isolated from cattle feces, since cattle are asymptomatic carriers of E. coli O104. The genomes of ten bovine E. coli O104 strains (six O104:H7, one O104:H8, one O104:H12, and two O104:H23) and five O104:H7 isolated from human clinical cases were sequenced. Of all the bovine O104 serotypes (H7, H8, H12, and H23) that were included in the study, only E. coli O104:H7 serotype possessed Shiga toxins. Four of the six bovine O104:H7 strains and one of the five human strains carried stx1c. Three human O104 strains carried stx2, two were of subtype 2a, and one was 2d. Genomes of stx carrying bovine O104:H7 strains were larger than the stx-negative strains of O104:H7 or other serotypes. The genome sizes were proportional to the number of genes carried on the mobile genetic elements (phages, prophages, transposable elements and plasmids). Both bovine and human strains were negative for intimin and other genes associated with the type III secretory system and non-LEE encoded effectors. Plasmid-encoded virulence genes (ehxA, epeA, espP, katP) were also present in bovine and human strains. All O104 strains were negative for antimicrobial resistance genes, except one human strain. Phylogenetic analysis indicated that bovine E. coli O104 strains carrying the same flagellar antigen clustered together and STEC strains clustered separately from non-STEC strains. One of the human O104:H7 strains was phylogenetically closely related to and belonged to the same sequence type (ST-1817) as the bovine O104:H7 STEC strains. This suggests that the bovine feces could be a source of human illness caused by E. coli O104:H7 serotype. Because bovine O104:H7 strains carried virulence genes similar to human clinical strains and one of the human clinical strains was phylogenetically related to bovine strains, the serotype has the potential to be a diarrheagenic pathogen in humans.

Draft Genome Sequences of 72 Isolates from All Four Species of Shigella.

Shigella is a genus of Gram-negative enteric pathogenic bacteria which has four species, Shigella dysenteriae, S. flexneri, S. boydii, and S. sonnei. Shigella species are clinically important bacteria because they cause shigellosis or dysentery. Here we report the genome sequences of 72 Shigella isolates from these four species.

DNA microarray-based assessment of virulence potential of Shiga toxin gene-carrying Escherichia coli O104:H7 isolated from feedlot cattle feces.

Escherichia coli O104:H4, a hybrid pathotype reported in a large 2011 foodborne outbreak in Germany, has not been detected in cattle feces. However, cattle harbor and shed in the feces other O104 serotypes, particularly O104:H7, which has been associated with sporadic cases of diarrhea in humans. The objective of our study was to assess the virulence potential of Shiga toxin-producing E. coli (STEC) O104:H7 isolated from feces of feedlot cattle using DNA microarray. Six strains of STEC O104:H7 isolated from cattle feces were analyzed using FDA-E. coli Identification (ECID) DNA microarray to determine their virulence profiles and compare them to the human strains (clinical) of O104:H7, STEC O104:H4 (German outbreak strain), and O104:H21 (milk-associated Montana outbreak strain). Scatter plots were generated from the array data to visualize the gene-level differences between bovine and human O104 strains, and Pearson correlation coefficients (r) were determined. Splits tree was generated to analyze relatedness between the strains. All O104:H7 strains, both bovine and human, similar to O104:H4 and O104:H21 outbreak strains were negative for intimin (eae). The bovine strains were positive for Shiga toxin 1 subtype c (stx1c), enterohemolysin (ehxA), tellurite resistance gene (terD), IrgA homolog protein (iha), type 1 fimbriae (fimH), and negative for genes that code for effector proteins of type III secretory system. The six cattle O104 strains were closely related (r = 0.86-0.98) to each other, except for a few differences in phage related and non-annotated genes. One of the human clinical O104:H7 strains (2011C-3665) was more closely related to the bovine O104:H7 strains (r = 0.81-0.85) than the other four human clinical O104:H7 strains (r = 0.75-0.79). Montana outbreak strain (O104:H21) was more closely related to four of the human clinical O104:H7 strains than the bovine O104:H7 strains. None of the bovine E. coli O104 strains carried genes characteristic of E. coli O104:H4 German outbreak strain and unlike other human strains were also negative for Shiga toxin 2. Because cattle E. coli O104:H7 strains possess stx1c and genes that code for enterohemolysin and a variety of adhesins, the serotype has the potential to be a diarrheagenic foodborne pathogen in humans.