Strain and host-cell dependent role of type-1 fimbriae in the adherence phenotype of super-shed Escherichia coli O157:H7.

Super-shed (SS) Escherichia coli O157 (E. coli O157) demonstrate a strong, aggregative, locus of enterocyte effacement (LEE)-independent adherence phenotype on bovine recto-anal junction squamous epithelial (RSE) cells, and harbor polymorphisms in non-LEE-adherence-related loci, including in the type 1 fimbriae operon. To elucidate the role of type 1 fimbriae in strain- and host-specific adherence, we evaluated the entire Fim operon (FimB-H) and its adhesion (FimH) deletion mutants in four E. coli O157 strains, SS17, SS52, SS77 and EDL933, and evaluated the adherence phenotype in bovine RSE and human HEp-2 adherence assays. Consistent with the prevailing dogma that fimH expression is genetically switched off in E. coli O157, the ΔfimHSS52, ΔfimB-HSS52, ΔfimB-HSS17, and ΔfimHSS77 mutants remained unchanged in adherence phenotype to RSE cells. In contrast, the ΔfimHSS17 and ΔfimB-HSS77 mutants changed from a wild-type strong and aggregative, to a moderate and diffuse adherence phenotype, while both ΔfimHEDL933 and ΔfimB-HEDL933 mutants demonstrated enhanced binding to RSE cells (p < 0.05). Additionally, both ΔfimHSS17 and ΔfimHEDL933 were non-adherent to HEp-2 cells (p < 0.05). Complementation of the mutant strains with their respective wild-type genes restored parental phenotypes. Microscopy revealed that the SS17 and EDL933 strains indeed carry type 1 fimbriae-like structures shorter than those seen in uropathogenic E. coli. Taken together, these results provide compelling evidence for a strain and host cell type-dependent role of fimH and the fim operon in E. coli O157 adherence that needs to be further evaluated.

Detection of the top six non-O157 Shiga toxin-producing Escherichia coli O groups by ELISA.

There is a growing concern of a public health risk associated with non-O157 Shiga toxin-producing Escherichia coli (STEC) since E. coli serogroups O26, O45, O103, O111, O121, and O145 are frequently implicated in outbreaks of human illness worldwide. Recently, the Food Safety and Inspection Service of the U.S. Department of Agriculture declared these six STEC O groups to be adulterants in beef. We describe here a rapid, sensitive, and highly specific enzyme-linked immunosorbent assay (ELISA) for the detection of these top six non-O157 STEC O groups. The assays were tested against 174 reference E. coli O groups, with 60 clinical isolates belonging to the target O groups and 10 non-E coli strains belonging to the family Enterobacteriaceae. Assays for serogroups O103, O111, and O121 exhibited 100% specificity, while assays for serogroups O26 and O45 had 98.2% specificity, and O145 had 99.1% specificity. ELISA conducted using artificially inoculated ground beef samples displayed 100% accuracy. The sensitivity of the assay was 5×10(5) colony-forming unit (CFU)/mL, with limits of detection in the range of 1-10 CFU/25 g of ground beef sample following enrichment. The findings of the study suggest that the assay described is simple and rapid, and can be employed to detect target STEC O groups in beef and other food samples. In addition, the assay provides a conceptual framework that can be adapted for the development of similar tests for the rapid detection of other serogroups of E. coli.

Nonfimbrial Adhesin Mutants Reveal Divergent Escherichia coli O157:H7 Adherence Mechanisms on Human and Cattle Epithelial Cells.

Shiga toxin-producing, enterohemorrhagic Escherichia coli (EHEC) serotype O157:H7 is a major foodborne pathogen causing symptoms ranging from simple intestinal discomfort to bloody diarrhea and life-threatening hemolytic uremic syndrome in humans. Cattle can be asymptomatically colonized by O157:H7 predominantly at the rectoanal junction (RAJ). Colonization of the RAJ is highly associated with the shedding of O157:H7 in bovine feces. Supershedding (SS) is a phenomenon that has been reported in some cattle that shed more than 104 colony-forming units of O57:H7 per gram of feces, 100-1000 times more or greater than normal shedders. The unique bovine RAJ cell adherence model revealed that O157:H7 employs a LEE-independent mechanism of attachment to one of the RAJ cell types, the squamous epithelial (RSE) cells. Nine nonfimbrial adhesins were selected to determine their role in the characteristic hyperadherent phenotype of SS O157 on bovine RSE cells, in comparison with human HEp-2 cells. A number of single nucleotide polymorphisms (SNPs) were found amongst these nonfimbrial adhesins across a number of SS isolates. In human cells, deletion of yfaL reduced the adherence of both EDL933 and SS17. However, deletion of eae resulted in a significant loss of adherence in SS17 whereas deletion of wzzB and iha in EDL933 resulted in the same loss of adherence to HEp-2 cells. On RSE cells, none of these nonfimbrial deletion mutants were able to alter the adherence phenotype of SS17. In EDL933, deletion of cah resulted in mitigated adherence. Surprisingly, four nonfimbrial adhesin gene deletions were actually able to confer the hyperadherent phenotype on RSE cells. Overall, this study reveals that the contribution of nonfimbrial adhesins to the adherence mechanisms and functions of O157:H7 is both strain and host cell type dependent as well as indicates a possible role of these nonfimbrial adhesins in the SS phenotype exhibited on RSE cells.

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.

Complete Genome Sequence of SS52, a Strain of Escherichia coli O157:H7 Recovered from Supershedder Cattle.

Shiga toxin-producing Escherichia coli O157:H7 causes foodborne infections, and cattle are the primary reservoir. Some animals, known as supershedders, excrete orders of magnitude more E. coli O157:H7 in the feces than normal. Here, we report the complete genome sequence of the SS52 supershedder strain of E. coli O157:H7.

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.

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.

Complete Genome Sequence of Mycobacterium avium subsp. paratuberculosis, Isolated from Human Breast Milk.

Mycobacterium avium subsp. paratuberculosis is the etiologic agent of Johne's disease in ruminants and has also been associated with human Crohn's disease. We report the complete genome sequence of M. avium subsp. paratuberculosis, isolated from the breast milk of a Crohn's disease patient. This sequence has high identity with characterized strains recovered from cattle.