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

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.

Curli Temper Adherence of Escherichia coli O157:H7 to Squamous Epithelial Cells from the Bovine Recto-Anal Junction in a Strain-Dependent Manner

“…In addition, all SS-O157 and control O157 strains adhered to HEp-2 cells in a "diffuse and moderate" pattern (Table 1). An initial screening on CRI plates confirmed that the control strains were curli-expressing (C ϩ ) strains as reported in other studies (27), but the SS-O157 strains differed in their levels of curli production, with only strains SS-12 and SS-42 appearing to be C ϩ (34,35). Since neither the RSE cell adherence patterns nor the HEp-2 cell adherence patterns of these O157 strains could be associated with their respective curli phenotype (Table 1), it appeared that, in contrast to previous reports (25)(26)(27)29), the adherence was not dependent on the curli fimbriae.…”

“…Eight previously characterized O157 isolates obtained from supershedding cattle were used in this study (34,35). These supershed (SS) O157 isolates included SS-7, SS-12, SS-17, SS-27, SS-42, SS-52, SS-67, and SS-77 (Table 1) (34,35).…”

“…These supershed (SS) O157 isolates included SS-7, SS-12, SS-17, SS-27, SS-42, SS-52, SS-67, and SS-77 (Table 1) (34,35). In addition, O157 strains EDL933, a sequenced human clinical isolate (46), 86-24, a clinical isolate (86-24 wild type, 86-24WT) (47), and its streptomycin-resistant derivative 86-24Sm r (48) were included as controls (Table 1).…”

“…Curli variants were derived from two O157 strains expressing only Shiga toxin 2 (Stx2) with contrasting adherence patterns on RSE cells and differences in curli production (this study), SS-17 and 86-24WT, using a previously described protocol (Table 2) (30,(33)(34)(35)47). Strain SS-17, demonstrating aggregative, strong adherence on RSE cells, was identified as curli negative (this study and references 34 and 35) upon initial screening.…”

“…tive 86-24Sm r (Table 1) (22,35). In addition, all SS-O157 and control O157 strains adhered to HEp-2 cells in a "diffuse and moderate" pattern (Table 1).…”