Comparative genomics of two super-shedder isolates of Escherichia coli O157:H7

Shiga toxin-producing Escherichia coli O157:H7 (O157) are zoonotic foodborne pathogens and of major public health concern that cause considerable intestinal and extra-intestinal illnesses in humans. O157 colonize the recto-anal junction (RAJ) of asymptomatic cattle who shed the bacterium into the environment through fecal matter. A small subset of cattle, termed super-shedders (SS), excrete O157 at a rate (104 CFU/g of feces) that is several orders of magnitude greater than other colonized cattle and play a major role in the prevalence and transmission of O157. To better understand microbial factors contributing to super-shedding we have recently sequenced two SS isolates, SS17 (GenBank accession no. CP008805) and SS52 (GenBank accession no. CP010304) and shown that SS isolates display a distinctive strongly adherent phenotype on bovine rectal squamous epithelial cells. Here we present a detailed comparative genomics analysis of SS17 and SS52 with other previously characterized O157 strains (EC4115, EDL933, Sakai, TW14359). The results highlight specific polymorphisms and genomic features shared amongst SS strains, and reveal several SNPs that are shared amongst SS isolates, including in genes involved in motility, adherence, and metabolism. Finally, our analyses reveal distinctive patterns of distribution of phage-associated genes amongst the two SS and other isolates. Together, the results of our comparative genomics studies suggest that while SS17 and SS52 share genomic features with other lineage I/II isolates, they likely have distinct recent evolutionary histories. Future comparative and functional genomic studies are needed to decipher the precise molecular basis for super shedding in O157

Proteins other than the locus of enterocyte effacement-encoded proteins contribute to Escherichia coli O157:H7 adherence to bovine rectoanal junction stratified squamous epithelial cells

Background: In this study, we present evidence that proteins encoded by the Locus of Enterocyte Effacement (LEE), considered critical for Escherichia coli O157 (O157) adherence to follicle-associated epithelial (FAE) cells at the bovine recto-anal junction (RAJ), do not appear to contribute to O157 adherence to squamous epithelial (RSE) cells also constituting this primary site of O157 colonization in cattle. Results: Antisera targeting intimin-γ, the primary O157 adhesin, and other essential LEE proteins failed to block O157 adherence to RSE cells, when this pathogen was grown in DMEM, a culture medium that enhances expression of LEE proteins. In addition, RSE adherence of a DMEM-grown-O157 mutant lacking the intimin protein was comparable to that seen with its wild-type parent O157 strain grown in the same media. These adherence patterns were in complete contrast to that observed with HEp-2 cells (the adherence to which is mediated by intimin-γ), assayed under same conditions. This suggested that proteins other than intimin-γ that contribute to adherence to RSE cells are expressed by this pathogen during growth in DMEM. To identify such proteins, we defined the proteome of DMEM-grown-O157 (DMEM-proteome). GeLC-MS/MS revealed that the O157 DMEM-proteome comprised 684 proteins including several components of the cattle and human O157 immunome, orthologs of adhesins, hypothetical secreted and outer membrane proteins, in addition to the known virulence and LEE proteins. Bioinformatics-based analysis of the components of the O157 DMEM proteome revealed several new O157-specific proteins with adhesin potential. Conclusion: Proteins other than LEE and intimin-γ proteins are involved in O157 adherence to RSE cells at the bovine RAJ. Such proteins, with adhesin potential, are expressed by this human pathogen during growth in DMEM. Ongoing experiments to evaluate their role in RSE adherence should provide both valuable insights into the O157-RSE interactions and new targets for more efficacious anti-adhesion O157 vaccines

Polymorphic Amplified Typing Sequences (PATS) Strain Typing System Accurately Discriminates a Set of Temporally and Spatially Disparate Escherichia coli O157 Isolates Associated with Human Infection

Polymorphic Amplified Typing Sequences (PATS) is a PCR-based Escherichia coli O157 (O157) strain typing system. Here, we show that PATS compares excellently with Pulsed-Field Gel Electrophoresis (PFGE) in that both methods cluster geographically diverse O157 isolates similarly. Comparative analysis of the results obtained in this simulated “blind” study attests to the discriminating power and applicability of PATS to epidemiological/nosocomial situations

AB₅ Enterotoxin-Mediated Pathogenesis: Perspectives Gleaned from Shiga Toxins

Foodborne diseases affect an estimated 600 million people worldwide annually, with the majority of these illnesses caused by Norovirus, Vibrio, Listeria, Campylobacter, Salmonella, and Escherichia coli. To elicit infections in humans, bacterial pathogens express a combination of virulence factors and toxins. AB5 toxins are an example of such toxins that can cause various clinical manifestations, including dehydration, diarrhea, kidney damage, hemorrhagic colitis, and hemolytic uremic syndrome (HUS). Treatment of most bacterial foodborne illnesses consists of fluid replacement and antibiotics. However, antibiotics are not recommended for infections caused by Shiga toxin-producing E. coli (STEC) because of the increased risk of HUS development, although there are conflicting views and results in this regard. Lack of effective treatment strategies for STEC infections pose a public health threat during outbreaks; therefore, the debate on antibiotic use for STEC infections could be further explored, along with investigations into antibiotic alternatives. The overall goal of this review is to provide a succinct summary on the mechanisms of action and the pathogenesis of AB5 and related toxins, as expressed by bacterial foodborne pathogens, with a primary focus on Shiga toxins (Stx). The role of Stx in human STEC disease, detection methodologies, and available treatment options are also briefly discussed

The Escherichia coli O157:H7 carbon starvation-inducible lipoprotein Slp contributes to initial adherence in vitro via the human polymeric immunoglobulin receptor.

Escherichia coli O157:H7 is the most well-studied serotype of the enterohemorrhagic E. coli (EHEC) class of E. coli intestinal pathogens and is responsible for many outbreaks of serious food-borne illness worldwide each year. Adherence mechanisms are a critical component of its pathogenesis, persistence in natural reservoirs, and environmental contamination. E. coli O157:H7 has a highly effective virulence operon, the Locus of Enterocyte Effacement (LEE), and its encoded intimate adherence mechanism is well characterized. However, factors involved in the preceding initial attachment are not well understood. In this study, we propose a mechanism of initial adherence used by E. coli O157:H7 in vitro. We describe a bacterial protein not previously reported to be involved in adherence, Slp, and its interactions with the human host protein polymeric immunoglobulin receptor (pIgR). The human pIgR has previously been shown to act as an adherence receptor for some mucosal pathogens and is highly expressed in the intestine. Following observation of significant colocalization between E. coli O157:H7 bacteria and pIgR location on Caco-2 cells, a co-immunoprecipitation (Co-IP) assay using a human recombinant Fc-tagged pIgR protein led to the identification of this protein. Disruption of Slp expression in E. coli O157:H7, through deletion of its encoding gene slp, produced a significant adherence deficiency to Caco-2 cells at early time points associated with initial adherence. Plasmid complementation of the slp gene fully restored the wild-type phenotype. Furthermore, immunofluorescence microscopy revealed evidence that this interaction is specific to the pathogenic strains of E. coli tested and not the nonpathogenic control strain E. coli K12. Additionally, deletion of slp gene resulted in the absence of the corresponding protein band in further Co-IP assays, while the plasmid-encoded slp gene complementation of the deletion mutant strain restored the wild-type pattern. These data support the proposal that Slp directly contributes to initial adherence, with the pIgR protein as its proposed receptor

Comparative genomics of two super-shedder isolates of Escherichia coli O157:H7

Shiga toxin-producing Escherichia coli O157:H7 (O157) are zoonotic foodborne pathogens and of major public health concern that cause considerable intestinal and extra-intestinal illnesses in humans. O157 colonize the recto-anal junction (RAJ) of asymptomatic cattle who shed the bacterium into the environment through fecal matter. A small subset of cattle, termed super-shedders (SS), excrete O157 at a rate (104 CFU/g of feces) that is several orders of magnitude greater than other colonized cattle and play a major role in the prevalence and transmission of O157. To better understand microbial factors contributing to super-shedding we have recently sequenced two SS isolates, SS17 (GenBank accession no. CP008805) and SS52 (GenBank accession no. CP010304) and shown that SS isolates display a distinctive strongly adherent phenotype on bovine rectal squamous epithelial cells. Here we present a detailed comparative genomics analysis of SS17 and SS52 with other previously characterized O157 strains (EC4115, EDL933, Sakai, TW14359). The results highlight specific polymorphisms and genomic features shared amongst SS strains, and reveal several SNPs that are shared amongst SS isolates, including in genes involved in motility, adherence, and metabolism. Finally, our analyses reveal distinctive patterns of distribution of phage-associated genes amongst the two SS and other isolates. Together, the results of our comparative genomics studies suggest that while SS17 and SS52 share genomic features with other lineage I/II isolates, they likely have distinct recent evolutionary histories. Future comparative and functional genomic studies are needed to decipher the precise molecular basis for super shedding in O157

Application of Bacteriophages To Control Intestinal Escherichia coli O157:H7 Levels in Ruminants

A previously characterized O157-specific lytic bacteriophage KH1 and a newly isolated phage designated SH1 were tested, alone or in combination, for reducing intestinal Escherichia coli O157:H7 in animals. Oral treatment with phage KH1 did not reduce the intestinal E. coli O157:H7 in sheep. Phage SH1 formed clear and relatively larger plaques on lawns of all 12 E. coli O157:H7 isolates tested and had a broader host range than phage KH1, lysing O55:H6 and 18 of 120 non-O157 E. coli isolates tested. In vitro, mucin or bovine mucus did not inhibit bacterial lysis by phage SH1 or KH1. A phage treatment protocol was optimized using a mouse model of E. coli O157:H7 intestinal carriage. Oral treatment with SH1 or a mixture of SH1 and KH1 at phage/bacterium ratios ≥10(2) terminated the presence of fecal E. coli O157:H7 within 2 to 6 days after phage treatment. Untreated control mice remained culture positive for >10 days. To optimize bacterial carriage and phage delivery in cattle, E. coli O157:H7 was applied rectally to Holstein steers 7 days before the administration of 10(10) PFU SH1 and KH1. Phages were applied directly to the rectoanal junction mucosa at phage/bacterium ratios calculated to be ≥10(2). In addition, phages were maintained at 10(6) PFU/ml in the drinking water of the phage treatment group. This phage therapy reduced the average number of E. coli O157:H7 CFU among phage-treated steers compared to control steers (P < 0.05); however, it did not eliminate the bacteria from the majority of steers