ABSTRACT: As it descended from Escherichia coli O55:H7, Shiga toxin (Stx)-producing E. coli (STEC) O157:H7 is believed to have acquired, in sequence, a bacteriophage encoding Stx2 and another encoding Stx1. Between these events, sorbitol-fermenting E. coli O157:H(-) presumably diverged from this clade. We employed PCR and sequence analyses to investigate sites of bacteriophage integration into the chromosome, using evolutionarily informative STEC to trace the sequence of acquisition of elements encoding Stx. Contrary to expectations from the two currently sequenced strains, truncated bacteriophages occupy yehV in almost all E. coli O157:H7 strains that lack stx(1) (stx(1)-negative strains). Two truncated variants were determined to contain either GTT or TGACTGTT sequence, in lieu of 20,214 or 18,895 bp, respectively, of the bacteriophage central region. A single-nucleotide polymorphism in the latter variant suggests that recombination in that element extended beyond the inserted octamer. An stx(2) bacteriophage usually occupies wrbA in stx(1)(+)/stx(2)(+) E. coli O157:H7, but wrbA is unexpectedly unoccupied in most stx(1)-negative/stx(2)(+) E. coli O157:H7 strains, the presumed progenitors of stx(1)(+)/stx(2)(+) E. coli O157:H7. Trimethoprim-sulfamethoxazole promotes the excision of all, and ciprofloxacin and fosfomycin significantly promote the excision of a subset of complete and truncated stx bacteriophages from the E. coli O157:H7 strains tested; bile salts usually attenuate excision. These data demonstrate the unexpected diversity of the chromosomal architecture of E. coli O157:H7 (with novel truncated bacteriophages and multiple stx(2) bacteriophage insertion sites), suggest that stx(1) acquisition might be a multistep process, and compel the consideration of multiple exogenous factors, including antibiotics and bile, when chromosome stability is examined.
Project description:Shiga toxin (Stx) are cardinal virulence factors of enterohemorrhagic E. coli O157:H7 (EHEC O157). The gene content and genomic insertion sites of Stx-associated bacteriophages differentiate clinical genotypes of EHEC O157 (CG, typical of clinical isolates) from bovine-biased genotypes (BBG, rarely identified among clinical isolates). This project was designed to identify bacteriophage-mediated differences that may affect the virulence of CG and BBG.Stx-associated bacteriophage differences were identified by whole genome optical scans and characterized among >400 EHEC O157 clinical and cattle isolates by PCR.Optical restriction maps of BBG strains consistently differed from those of CG strains only in the chromosomal insertion sites of Stx2-associated bacteriophages. Multiplex PCRs (stx1, stx2a, and stx2c as well as Stx-associated bacteriophage-chromosomal insertion site junctions) revealed four CG and three BBG that accounted for >90% of isolates. All BBG contained stx2c and Stx2c-associated bacteriophage-sbcB junctions. All CG contained stx2a and Stx2a-associated bacteriophage junctions in wrbA or argW.Presence or absence of stx2a (or another product encoded by the Stx2a-associated bacteriophage) is a parsimonious explanation for differential virulence of BBG and CG, as reflected in the distributions of these genotypes in humans and in the cattle reservoir.
Project description:Shiga toxin-converting bacteriophages are involved in the pathogenicity of some enteric bacteria, such as Escherichia coli O157:H7, but data on the occurrence and distribution of such phages as free particles in nature were not available. An experimental approach has been developed to detect the presence of the Shiga toxin 2 (Stx 2)-encoding bacteriophages in sewage. The Stx 2 gene was amplified by PCR from phages concentrated from 10-ml samples of sewage. Moreover, the phages carrying the Stx 2 gene were detected in supernatants from bacteriophage enrichment cultures by using an Stx 2-negative E. coli O157:H7 strain infected with phages purified from volumes of sewage as small as 0.02 ml. Additionally, the A subunit of Stx 2 was detected in the supernatants of the bacteriophage enrichment cultures, which also showed cytotoxic activity for Vero cells. By enrichment of phages concentrated from different volumes of sewage and applying the most-probable-number technique, it was estimated that the number of phages infectious for E. coli O157:H7 and carrying the Stx 2 gene was in the range of 1 to 10 per ml of sewage from two different origins. These values were approximately 1% of all phages infecting E. coli O157:H7.
Project description:Escherichia coli O157:H7, a zoonotic human pathogen for which domestic cattle are a reservoir host, produces a Shiga toxin(s) (Stx) encoded by bacteriophages. Chromosomal insertion sites of these bacteriophages define three principal genotypes (clusters 1 to 3) among clinical isolates of E. coli O157:H7. Stx-encoding bacteriophage insertion site genotypes of 282 clinical and 80 bovine isolates were evaluated. A total of 268 (95.0%) of the clinical isolates, but only 41 (51.3%) of the bovine isolates, belonged to cluster 1, 2, or 3 (P < 0.001). Thirteen additional genotypes were identified in isolates from both cattle and humans (four genotypes), from only cattle (seven genotypes), or from only humans (two genotypes). Two other markers previously associated with isolates from cattle or with clinical isolates showed similar associations with genotype groups within bovine isolates; the tir allele sp-1 and the Q933W allele were under- and overrepresented, respectively, among cluster 1 to 3 genotypes. Stx-encoding bacteriophage insertion site typing demonstrated that there is broad genetic diversity of E. coli O157:H7 in the bovine reservoir and that numerous genotypes are significantly underrepresented among clinical isolates, consistent with the possibility that there is reduced virulence or transmissibility to humans of some bovine E. coli O157:H7 genotypes.
Project description:Enteropathogenic Escherichia coli (EPEC) continues to be a leading cause of mortality and morbidity in children around the world. Two EPEC genomes have been fully sequenced: those of EPEC O127:H6 strain E2348/69 (United Kingdom, 1969) and EPEC O55:H7 strain CB9615 (Germany, 2003). The O55:H7 serotype is a recent precursor to the virulent enterohemorrhagic E. coli O157:H7. To explore the diversity of O55:H7 and better understand the clonal evolution of O157:H7, we fully sequenced EPEC O55:H7 strain RM12579 (California, 1974), which was collected 1 year before the first U.S. isolate of O157:H7 was identified in California. Phage-related sequences accounted for nearly all differences between the two O55:H7 strains. Additionally, O55:H7 and O157:H7 strains were tested for the presence and insertion sites of Shiga toxin gene (stx)-containing bacteriophages. Analysis of non-phage-associated genes supported core elements of previous O157:H7 stepwise evolutionary models, whereas phage composition and insertion analyses suggested a key refinement. Specifically, the placement and presence of lambda-like bacteriophages (including those containing stx) should not be considered stable evolutionary markers or be required in placing O55:H7 and O157:H7 strains within the stepwise evolutionary models. Additionally, we suggest that a 10.9-kb region (block 172) previously believed unique to O55:H7 strains can be used to identify early O157:H7 strains. Finally, we defined two subsets of O55:H7 strains that share an as-yet-unobserved or extinct common ancestor with O157:H7 strains. Exploration of O55:H7 diversity improved our understanding of the evolution of E. coli O157:H7 and suggested a key revision to accommodate existing and future configurations of stx-containing bacteriophages into current models.
Project description:Escherichia coli O157:H7 is a food-borne bacterium that causes hemorrhagic diarrhea and hemolytic uremic syndrome in humans. While cattle are a known source of E. coli O157:H7 exposure resulting in human infection, environmental reservoirs may also be important sources of infection for both cattle and humans. Bacteriophage-encoded Shiga toxins (Stx) carried by E. coli O157:H7 may provide a selective advantage for survival of these bacteria in the environment, possibly through their toxic effects on grazing protozoa. To determine Stx effects on protozoan grazing, we co-cultured Paramecium caudatum, a common ciliate protozoon in cattle water sources, with multiple strains of Shiga-toxigenic E. coli O157:H7 and non-Shiga toxigenic cattle commensal E. coli. Over three days at ambient laboratory temperature, P. caudatum consistently reduced both E. coli O157:H7 and non-Shiga toxigenic E. coli populations by 1-3 log cfu. Furthermore, a wild-type strain of Shiga-toxigenic E. coli O157:H7 (EDL933) and isogenic mutants lacking the A subunit of Stx 2a, the entire Stx 2a-encoding bacteriophage, and/or the entire Stx 1-encoding bacteriophage were grazed with similar efficacy by both P. caudatum and Tetrahymena pyriformis (another ciliate protozoon). Therefore, our data provided no evidence of a protective effect of either Stx or the products of other bacteriophage genes on protozoan predation of E. coli. Further research is necessary to determine if the grazing activity of naturally-occurring protozoa in cattle water troughs can serve to decrease cattle exposure to E. coli O157:H7 and other Shiga-toxigenic E. coli.
Project description:We used whole-genome sequencing to investigate the evolutionary context of an emerging highly pathogenic strain of Shiga toxin-producing Escherichia coli (STEC) O157:H7 in England and Wales. A timed phylogeny of sublineage IIb revealed that the emerging clone evolved from a STEC O157:H7 stx-negative ancestor ?10 years ago after acquisition of a bacteriophage encoding Shiga toxin (stx) 2a, which in turn had evolved from a stx2c progenitor ?20 years ago. Infection with the stx2a clone was a significant risk factor for bloody diarrhea (OR 4.61, 95% CI 2.24-9.48; p<0.001), compared with infection with other strains within sublineage IIb. Clinical symptoms of cases infected with sublineage IIb stx2c and stx-negative clones were comparable, despite the loss of stx2c. Our analysis highlighted the highly dynamic nature of STEC O157:H7 Stx-encoding bacteriophages and revealed the evolutionary history of a highly pathogenic clone emerging within sublineage IIb, a sublineage not previously associated with severe clinical symptoms.
Project description:The toxigenic conversion of Escherichia coli strains by Shiga toxin-converting (Stx) bacteriophages were prominent and recurring events in the stepwise evolution of enterohemorrhagic E. coli (EHEC) O157:H7 from an enteropathogenic (EPEC) O55:H7 ancestor. Atypical, attenuated isolates have been described for both non-sorbitol fermenting (NSF) O157:H7 and SF O157:NM serotypes, which are distinguished by the absence of Stx, the characteristic virulence hallmark of Stx-producing E. coli (STEC). Such atypical isolates either never acquired Stx-phages or may have secondarily lost stx during the course of infection, isolation, or routine subculture; the latter are commonly referred to as LST (Lost Shiga Toxin)-isolates. In this study we analyzed the genomes of 15 NSF O157:H7 and SF O157:NM strains from North America, Europe, and Asia that are characterized by the absence of stx, the virulence hallmark of STEC. The individual genomic basis of the Stx (-) phenotype has remained largely undetermined as the majority of STEC genomes in public genome repositories were generated using short read technology and are in draft stage, posing a major obstacle for the high-resolution whole genome sequence typing (WGST). The application of LRT (long-read technology) sequencing provided us with closed genomes, which proved critical to put the atypical non-shigatoxigenic NSF O157:H7 and SF O157:NM strains into the phylogenomic context of the stepwise evolutionary model. Availability of closed chromosomes for representative Stx (-) NSF O157:H7 and SF O157:NM strains allowed to describe the genomic basis and individual evolutionary trajectories underlying the absence of Stx at high accuracy and resolution. The ability of LRT to recover and accurately assemble plasmids revealed a strong correlation between the strains' featured plasmid genotype and chromosomally inferred clade, which suggests the coevolution of the chromosome and accessory plasmids. The identified ancestral traits in the pSFO157 plasmid of NSF O157:H7 strain LSU-61 provided additional evidence for its intermediate status. Taken together, these observations highlight the utility of LRTs for advancing our understanding of EHEC O157:H7/NM pathogenome evolution. Insights into the genomic and phenotypic plasticity of STEC on a lineage- and genome-wide scale are foundational to improve and inform risk assessment, biosurveillance, and prevention strategies.
Project description:Stx bacteriophages in 68 samples of beef and salad were quantified by real-time quantitative PCR (qPCR). Stx phages from the samples were propagated in Escherichia coli C600, E. coli O157:H7, and Shigella strains and further quantified. Fifty percent of the samples carried infectious Stx phages that were isolated from plaques generated by lysis.
Project description:The production of Shiga toxin (Stx) (verocytotoxin) is a major virulence factor of Escherichia coli O157:H7 strains (Shiga toxin-producing E. coli [STEC] O157). Two types of Shiga toxins, designated Stx1 and Stx2, are produced in STEC O157. Variants of the Stx2 type (Stx2, Stx2c) are associated with high virulences of these strains for humans. A bacteriophage designated 2851 from a human STEC O157 encoding the Stx2c variant was described previously. Nucleotide sequence analysis of the phage 2851 genome revealed 75 predicted coding sequences and indicated a mosaic structure typical for lambdoid phages. Analyses of free phages and K-12 phage 2851 lysogens revealed that upon excision from the bacterial chromosome, the loss of a phage-encoded IS629 element leads to fusion of phage antA and antB genes, with the generation of a recombined antAB gene encoding a strong antirepressor. In wild-type E. coli O157 as well as in K-12 strains, phage 2851 was found to be integrated in the sbcB locus. Additionally, phage 2851 carries an open reading frame which encodes an OspB-like type III effector similar to that found in Shigella spp. Investigation of 39 stx(2c) E. coli O157 strains revealed that all except 1 were positive for most phage 2851-specific genes and possessed a prophage with the same border sequences integrated into the sbcB locus. Phage 2851-specific sequences were absent from most stx(2c)-negative E. coli O157 strains, and we suggest that phage 2851-like phages contributed significantly to the dissemination of the Stx2c variant toxin within this group of E. coli.
Project description:Escherichia coli O157:H7 is a human pathogen that resides asymptomatically in its bovine host. The level of Shiga toxin (Stx) produced is variable in bovine-derived strains in contrast to human isolates that mostly produce high levels of Stx. To understand the genetic basis for varied Stx production, chronological collections of bovine isolates from Wisconsin dairy farms, R and X, were analyzed for multilocus prophage polymorphisms, stx(2) subtypes, and the levels of stx(2) transcript and toxin. The E. coli O157:H7 that persisted on both farms were phylogenetically distinct and yet produced little to no Stx2 due to gene deletions in Stx2c-encoding prophage (farm R) or insertional inactivation of stx(2a) by IS1203v (farm X). Loss of key regulatory and lysis genes in Stx2c-encoding prophage abolished stx(2c) transcription and induction of the prophage and stx(2a)::IS1203v in Stx2a-encoding prophage generated a truncated stx(2a) mRNA without affecting phage production. Stx2-producing strains were transiently present (farm R) and became Stx2 negative on farm X (i.e., stx(2a)::IS1203v). To our knowledge, this is the first study that details the evolution of E. coli O157:H7 and its Stx2-encoding prophage in a chronological collection of natural isolates. The data suggest the bovine and farm environments can be niches where Stx2-negative E. coli O157:H7 emerge and persist, which explains the Stx variability in bovine isolates and may be part of an evolutionary step toward becoming bovine specialists.