Genomic and Molecular Analysis of the Unique Phenotypes of an E. coli O157:h7 Super Shedder Isolate

Open Access
Author:
Moreau, Matthew R
Graduate Program:
Pathobiology
Degree:
Master of Science
Document Type:
Master Thesis
Date of Defense:
July 10, 2013
Committee Members:
  • Vivek Kapur, Thesis Advisor
  • Edward G Dudley, Thesis Advisor
  • Subhashinie Kariyawasam, Thesis Advisor
Keywords:
  • E. coli
  • Pathogen
  • O157:H7
  • EHEC
  • Biofilms
  • Reservoir
  • Super Shedder
  • Cow
  • Leafy Greens
  • Comparative Genomics
Abstract:
Shiga toxin producing Escherichia coli (STEC) are a subtype of pathogenic E. coli, and recently STEC serotype O157:H7 (EHEC O157:H7) have been recognized as a major foodborne pathogen causing symptoms ranging from having simple intestinal discomfort to bloody diarrhea and life threatening hemolytic uremic syndrome. Asymptomatic cattle are colonized with EHEC O157:H7 at the mucosal interface of the recto-anal junction (RAJ), and coincidentally this colonization of the RAJ is critical for the ability of this organism to be shed from the cattle. Super shedding (SS) is a phenomenon that has been reported in some cattle that shed EHEC O157:H7 at a rate greater than 104 colony forming units per gram of feces (CFU/g), 100-1000 times more or greater than normal shedders. The critical nature of colonization of the RAJ and shedding of EHEC O157:H7 indicated that this interaction might have been changed at a cellular level. The development of a unique cattle RAJ cell culture model revealed that O157:H7 employ a LEE-independent mechanism of attachment to the other RAJ cell type, recto-anal squamous epithelial (RSE) cells. SS isolates show a strong aggregative and adherence phenotype on RAJ cells, as opposed to two other O157:H7 strains, 86-24 and EDL933, which is also independent of the LEE operon. Other data show that these SS isolates have a higher affinity of binding leafy greens, such as lettuce and spinach, which is important because contaminated leafy greens serve as another major route of transmission of this pathogen. Since biofilms have been implicated in colonization and bacterial survival in a variety of environments, we investigated if SS17 could form biofilms, and the data indicate that SS17 has an enhanced ability to form biofilms compared to non-pathogenic E. coli strain K12 as well as EDL933 (a strain not believed to be an SS isolate). Recently, a representative SS isolate, SS17, was fully sequenced, and its genome was analyzed for changes in the genome (deletions and/or acquisition of novel genes) as well as single nucleotide polymorphisms (SNPs), focusing on virulence and adherence related genes. These analyses revealed that SS17 contained 295 virulence related genes, of which 39 had coding non-synonymous SNPs (nsSNPs). Of the 9 genes that were chosen through a rigorous selection process, none had a complete conservation of the same nsSNP in all 11 other SS isolates but were used to generate a phylogenetic analysis of the representative SS isolates. None of the genes chosen were shown to be individually essential for the change in adherence phenotype to the RSE cells seen in SS17; however, ompA, eivA, yfaL, and wzzB gene deletions in EDL933 were able to switch the phenotype in EDL933 from moderate aggregative to strong aggregative attachment seen in SS17. These data indicate that they may play a role in this phenotype but in an inhibitory manner and that a protein(s) they interact with may be involved directly. In SS17 it has now been shown that yfaL is critically important for the production of biofilm in context of leafy green extract; as is cah and ompA; but it is unclear if the nsSNPs exhibited by the two former genes have to do with the enhanced biofilm formation exhibited by SS17. All three show importance to the enhanced biofilm formation of SS17, and yfaL and ompA have for the first time been shown to be critical to biofilm formation in a strain of EHEC O157:H7; and all 3 important for SS17s ability to produce biofilms induced by factors released by damaged leaves.