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Comparative analysis of super-shedder strains of Escherichia coli O157:H7 reveals distinctive genomic features and a strongly aggregative adherent phenotype on bovine rectoanal junction squamous epithelial cells.

Cote R, Katani R, Moreau MR, Kudva IT, Arthur TM, DebRoy C, Mwangi MM, Albert I, Raygoza Garay JA, Li L, Brandl MT, Carter MQ, Kapur V - PLoS ONE (2015)

Bottom Line: A large number of non-synonymous SNPs and other polymorphisms were identified in SS17 as compared with other O157 strains (EC4115, EDL933, Sakai, TW14359), including in key adherence- and virulence-related loci.Molecular genetic and functional analyses of defined mutants of SS17 suggested that the strongly adherent aggregative phenotype amongst SS isolates is LEE-independent, and likely results from a novel mechanism.Taken together, our study provides a rational framework for investigating the molecular mechanisms associated with SS, and strong evidence that SS O157 isolates have distinctive features and use a LEE-independent mechanism for hyper-adherence to bovine rectal epithelial cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Veterinary and Biomedical Science, The Pennsylvania State University, University Park, Pennsylvania, United States of America; The Huck Institutes of the Life Sciences, The Pennsylvania State University, University Park, Pennsylvania, United States of America.

ABSTRACT
Shiga toxin-producing Escherichia coli O157:H7 (O157) are significant foodborne pathogens and pose a serious threat to public health worldwide. The major reservoirs of O157 are asymptomatic cattle which harbor the organism in the terminal recto-anal junction (RAJ). Some colonized animals, referred to as "super-shedders" (SS), are known to shed O157 in exceptionally large numbers (>104 CFU/g of feces). Recent studies suggest that SS cattle play a major role in the prevalence and transmission of O157, but little is known about the molecular mechanisms associated with super-shedding. Whole genome sequence analysis of an SS O157 strain (SS17) revealed a genome of 5,523,849 bp chromosome with 5,430 open reading frames and two plasmids, pO157 and pSS17, of 94,645 bp and 37,446 bp, respectively. Comparative analyses showed that SS17 is clustered with spinach-associated O157 outbreak strains, and belongs to the lineage I/II, clade 8, D group, and genotype 1, a subgroup of O157 with predicted hyper-virulence. A large number of non-synonymous SNPs and other polymorphisms were identified in SS17 as compared with other O157 strains (EC4115, EDL933, Sakai, TW14359), including in key adherence- and virulence-related loci. Phenotypic analyses revealed a distinctive and strongly adherent aggregative phenotype of SS17 on bovine RAJ stratified squamous epithelial (RSE) cells that was conserved amongst other SS isolates. Molecular genetic and functional analyses of defined mutants of SS17 suggested that the strongly adherent aggregative phenotype amongst SS isolates is LEE-independent, and likely results from a novel mechanism. Taken together, our study provides a rational framework for investigating the molecular mechanisms associated with SS, and strong evidence that SS O157 isolates have distinctive features and use a LEE-independent mechanism for hyper-adherence to bovine rectal epithelial cells.

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Stx2 induction in SS17 and Sakai strains.The levels of Stx2 produced by SS17 were unaffected by induction and showed higher levels of Stx2 in uninduced cultures when compared to Sakai.
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pone.0116743.g003: Stx2 induction in SS17 and Sakai strains.The levels of Stx2 produced by SS17 were unaffected by induction and showed higher levels of Stx2 in uninduced cultures when compared to Sakai.

Mentions: A total of 295 virulence related genes, which have been previously described in other O157 strains were identified in SS17 using an automated clusters of orthologous group (COG) classification together with manual curation (S3 Table) [44]. Many of these genes are involved in multiple functions via various pathogenesis mechanisms employed by O157 during infection, and thus are possible targets for the increased virulence seen in super-shedder isolates. Of these, 276 genes are in the chromosome and 19 are on the pO157 plasmid, and can be broadly divided into three distinct groups based on putative protein function (toxin, adherence, and other virulence-associated). The toxin group consists of 109 genes, including the 47 LEE encoded genes, the 35 non-LEE effectors, and the hemolysin genes, hlyABCD, encoded on pO157 [3, 45, 46]. In addition, SS17 encodes for the Shiga toxin (stx) genes stx2a (argW integration) and stx2c but lacks the stx1 gene. Similar to O157 strains EC4115 and TW14359, SS17 has two copies of stx2A, one located proximal to the stx2c variant, and the other located near stx2B. Unlike Sakai, SS17 has the ability to produce Shiga toxin 2 at high levels without induction, suggesting hypervirulence characteristics of this isolate (Fig. 3). Further investigation is required to evaluate the mechanism of this phenotype in relation to super-shedding. In addition to the stx genes found on the chromosome, pO157 encodes for the hemolysin genes, hlyABCD, which are important for survival of the pathogen in the host [45].


Comparative analysis of super-shedder strains of Escherichia coli O157:H7 reveals distinctive genomic features and a strongly aggregative adherent phenotype on bovine rectoanal junction squamous epithelial cells.

Cote R, Katani R, Moreau MR, Kudva IT, Arthur TM, DebRoy C, Mwangi MM, Albert I, Raygoza Garay JA, Li L, Brandl MT, Carter MQ, Kapur V - PLoS ONE (2015)

Stx2 induction in SS17 and Sakai strains.The levels of Stx2 produced by SS17 were unaffected by induction and showed higher levels of Stx2 in uninduced cultures when compared to Sakai.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4321836&req=5

pone.0116743.g003: Stx2 induction in SS17 and Sakai strains.The levels of Stx2 produced by SS17 were unaffected by induction and showed higher levels of Stx2 in uninduced cultures when compared to Sakai.
Mentions: A total of 295 virulence related genes, which have been previously described in other O157 strains were identified in SS17 using an automated clusters of orthologous group (COG) classification together with manual curation (S3 Table) [44]. Many of these genes are involved in multiple functions via various pathogenesis mechanisms employed by O157 during infection, and thus are possible targets for the increased virulence seen in super-shedder isolates. Of these, 276 genes are in the chromosome and 19 are on the pO157 plasmid, and can be broadly divided into three distinct groups based on putative protein function (toxin, adherence, and other virulence-associated). The toxin group consists of 109 genes, including the 47 LEE encoded genes, the 35 non-LEE effectors, and the hemolysin genes, hlyABCD, encoded on pO157 [3, 45, 46]. In addition, SS17 encodes for the Shiga toxin (stx) genes stx2a (argW integration) and stx2c but lacks the stx1 gene. Similar to O157 strains EC4115 and TW14359, SS17 has two copies of stx2A, one located proximal to the stx2c variant, and the other located near stx2B. Unlike Sakai, SS17 has the ability to produce Shiga toxin 2 at high levels without induction, suggesting hypervirulence characteristics of this isolate (Fig. 3). Further investigation is required to evaluate the mechanism of this phenotype in relation to super-shedding. In addition to the stx genes found on the chromosome, pO157 encodes for the hemolysin genes, hlyABCD, which are important for survival of the pathogen in the host [45].

Bottom Line: A large number of non-synonymous SNPs and other polymorphisms were identified in SS17 as compared with other O157 strains (EC4115, EDL933, Sakai, TW14359), including in key adherence- and virulence-related loci.Molecular genetic and functional analyses of defined mutants of SS17 suggested that the strongly adherent aggregative phenotype amongst SS isolates is LEE-independent, and likely results from a novel mechanism.Taken together, our study provides a rational framework for investigating the molecular mechanisms associated with SS, and strong evidence that SS O157 isolates have distinctive features and use a LEE-independent mechanism for hyper-adherence to bovine rectal epithelial cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Veterinary and Biomedical Science, The Pennsylvania State University, University Park, Pennsylvania, United States of America; The Huck Institutes of the Life Sciences, The Pennsylvania State University, University Park, Pennsylvania, United States of America.

ABSTRACT
Shiga toxin-producing Escherichia coli O157:H7 (O157) are significant foodborne pathogens and pose a serious threat to public health worldwide. The major reservoirs of O157 are asymptomatic cattle which harbor the organism in the terminal recto-anal junction (RAJ). Some colonized animals, referred to as "super-shedders" (SS), are known to shed O157 in exceptionally large numbers (>104 CFU/g of feces). Recent studies suggest that SS cattle play a major role in the prevalence and transmission of O157, but little is known about the molecular mechanisms associated with super-shedding. Whole genome sequence analysis of an SS O157 strain (SS17) revealed a genome of 5,523,849 bp chromosome with 5,430 open reading frames and two plasmids, pO157 and pSS17, of 94,645 bp and 37,446 bp, respectively. Comparative analyses showed that SS17 is clustered with spinach-associated O157 outbreak strains, and belongs to the lineage I/II, clade 8, D group, and genotype 1, a subgroup of O157 with predicted hyper-virulence. A large number of non-synonymous SNPs and other polymorphisms were identified in SS17 as compared with other O157 strains (EC4115, EDL933, Sakai, TW14359), including in key adherence- and virulence-related loci. Phenotypic analyses revealed a distinctive and strongly adherent aggregative phenotype of SS17 on bovine RAJ stratified squamous epithelial (RSE) cells that was conserved amongst other SS isolates. Molecular genetic and functional analyses of defined mutants of SS17 suggested that the strongly adherent aggregative phenotype amongst SS isolates is LEE-independent, and likely results from a novel mechanism. Taken together, our study provides a rational framework for investigating the molecular mechanisms associated with SS, and strong evidence that SS O157 isolates have distinctive features and use a LEE-independent mechanism for hyper-adherence to bovine rectal epithelial cells.

Show MeSH
Related in: MedlinePlus