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

Authors: COTE, REBECCA - Pennsylvania State University; KATANI, ROBAB - Pennsylvania State University; MATHEW, MOREAU - Pennsylvania State University; Kudva, Indira; Arthur, Terrance; DEBROY, CHITRITA - Pennsylvania State University; MWANGI, MICHAEL - Pennsylvania State University; ALBERT, ISTVAN - Pennsylvania State University; GARAY, JUAN-ANTONIO - Pennsylvania State University; LI, LINGLING - Pennsylvania State University; Brandl, Maria; Carter, Michelle; KAPUR, VIVEK - Pennsylvania State University

Submitted to: PLOS ONE
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/18/2014
Publication Date: 2/9/2015
Citation: Cote, R., Katani, R., Mathew, M.R., Kudva, I.T., Arthur, T.M., DebRoy, C., Mwangi, M.M., Albert, I., Garay, J.R., Li, L., Brandl, M., Carter, M.Q., Kapur, V. 2015. 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. PLoS One. 10(2):e0116743. DOI: 10.1371/journal.pone.0116743.

Interpretive Summary: Cattle are the primary source of Escherichia coli O157 (O157), a bacterium (micro-organism) responsible for bloody diarrhea and other serious symptoms in humans that can even lead to death. Although O157 can cause serious disease in humans, cattle carry this bacterium in their intestines without getting any disease and shed this bacterium in their feces. The cattle intestinal site at which O157 prefers to localize is called the recto-anal junction or RAJ that is found close to the anal canal. Some colonized animals, referred to as “super-shedders”, are known to shed O157 in exceptionally large numbers (greater than10**4 CFU/g of feces). Recent studies suggest that these supershedding cattle play a major role in the prevalence and transmission of O157, but little is known about the mechanisms causing this phenomenon. Three principle components, or a combination thereof, are considered likely to contribute to the super-shedding phenomenon and include, the microbe, the host and the environment. Understanding the roles of each of these components is important for the development of new methods to reduce/eliminate the high shedding and transmission of super-shed O157 isolates, and ultimately decreasing the food-borne illnesses associated with O157. While other researchers are evaluating the role of host and environment in the super-shedding phenomenon, we decided to take a closer look at the super-shed strains of O157. Our analysis of the complete genome of a super-shed O157 shows the presence of polymorphisms in important genes associated with adherence, toxicity and virulence that are not found in other non-super-shed O157 strains suggesting that these microbes may be uniquely evolved. We have also found that these super-shed O157 strains bind cells isolated from the RAJ in a strikingly strong, aggregative pattern compared to non-super-shed O157. These observations provide a rational framework upon which we plan to base our future investigations on the role of microbial factors in the super-shedding phenomenon.

Technical Abstract: Shiga toxin-producing Escherichia coli O157:H7 (O157) are significant foodborne pathogens and 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 (greater than 10**4 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 strain of O157 (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. Whole genome alignments and phylogenetic analyses revealed that SS17 clusters with previously described spinach outbreak strains of O157, and belongs to the lineage I/II, clade 8, D group, and genotype 1, group of isolates with a predicted hyper-virulent phenotype. A large number of non-synonymous SNPs and other polymorphisms were identified in SS17 as compared with previously characterized O157 strains (EC4115, EDL933, Sakai, TW14359), including in key adherence- and virulence- related loci. Phenotypic analyses of SS17 revealed a striking strongly adherent aggregative phenotype on bovine RAJ stratified squamous epithelial (RSE) cells conserved amongst SS isolates but distinct from the phenotype observed with previously characterized reference O157 strains. Subsequent molecular genetic analyses with defined mutants of SS17 together with functional neutralization assays revealed that the strongly adherent aggregative phenotype amongst SS isolates is Locus of Enterocyte Effacement (LEE) independent. Taken together, the results of our studies provide a rational framework for investigating the molecular mechanisms associated with SS, and strong evidence that SS isolates of O157 represent specialized variants of O157 that use a LEE independent mechanism for hyper-adherence to bovine rectal epithelial cells.