|Bono, James - Jim|
|Clawson, Michael - Mike|
|Heaton, Michael - Mike|
Submitted to: American Society of Microbiologists Abstracts
Publication Type: Abstract Only
Publication Acceptance Date: 3/7/2005
Publication Date: 6/1/2005
Citation: Bono, J.L., Keen, J.E., Clawson, M.L., Heaton, M.P., Chitko Mckown, C.G., Miller, L.C., Laegreid, W.W. 2005. Discrimintion of Escherichia coli O157:H7 isolates by genotyping single nucleotide polymorphisms. [abstract] American Society of Microbiologists. p. 318.
Technical Abstract: Escherichia coli O157:H7 is the major cause of hemorrhagic colitis and hemolytic uremic syndrome in the United States. The locus of enterocyte effacement (LEE) is one of the pathogenicity islands of E. coli O157:H7 which encodes for genes involved in the adherence of E. coli O157:H7 to intestinal epithelial cells. The adherence is characterized by attaching and effacing lesions, which causes destruction of the microvilli and the formation of pedestal-like structures. Two genes encoded in the LEE locus, tir and eae, are responsible for the tight association of the bacterium with the intestinal epithelial cell. The eae encoded protein, intimin, is located on the outside of the bacterium while the intimin receptor protein Tir is tranlocated into the epithelial cell by a type III secretion pathway where it is integrated in the cell membrane. Because the Tir protein is an important part of the adherence process any nucleotide variation within the tir gene could alter the receptor properties of this protein. Therefore, we hypothesized that genomic differences in the tir gene could categorize the pathogenicity of E. coli O157:H7 isolates in either humans or cattle. The tir gene was sequenced from 8 human and 12 bovine E. coli O157:H7 isolates. An 18 base pair insertion in the tir gene was found in a subset of the bovine isolates while the wild-type sequence was found in both human and bovine isolates. Alleles of a nucleotide polymorphism (tir-sp1) located 358 bases upstream of the indel were correlated with the indel alleles. A real-time PCR genotyping assay was designed for the tir-sp1 site and used to genotype 142 human and 68 bovine E. coli O157:H7 isolates, respectively. The distribution of genotypes differed between human and bovine isolates, with 1% of the human isolates and 35% of bovine isolates having the tir-sp1 indel allele (p< 0.000001). These results indicate that alleles at the tir-sp1 site may be useful markers for identifying a subset of E. coli O157:H7 that cause disease in humans.