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United States Department of Agriculture

Agricultural Research Service

Research Project: PREVENTION AND CONTROL OF SHIGA-TOXIGENIC ESCHERICHIA COLI IN LIVESTOCK Title: Discrimination of Escherichia Coli O157:h7 Isolates by Genotyping Single Nucleotide Polymorphisms

Authors
item Bono, James
item Keen, James
item Laegreid, William

Submitted to: International Symposium and Workshop on Shiga Toxin ... Escherichia coli
Publication Type: Abstract Only
Publication Acceptance Date: July 18, 2006
Publication Date: October 29, 2006
Citation: Bono, J.L., Keen, J.E., Laegreid, W.W. 2006. Discrimination of Escherichia coli O157:H7 isolates by genotyping single nucleotide polymorphisms [abstract]. Proceedings of the 6th International Symposium on Shiga Toxin (Verocytotoxin)--Producing Escherichia coli Infections, October 29 - November 1, 2006, Melbourne, Australia. Poster No. P13.1.07.

Technical Abstract: Current typing methods for enterohemorrhagic Escherichia coli (EHEC) O157:H7 include multilocus enzyme electrophoresis (MLEE), phage typing, plasmid profiling, random or arbitrarily primed polymerase chain reaction (RAP-PCR), and various forms of restriction fragment polymorphism analysis (RFLP). These methods suffer from a variety of problems including poor repeatability between laboratories, poor adaptability to automated analysis, lack of sufficient resolution to discriminate between unrelated isolates (false positive relatedness), and between truly related isolates (false negative relatedness). To type E. coli O157:H7 isolates, we sequenced targeted loci in the E. coli O157:H7 genome from a diverse panel of 24 E. coli O157:H7 isolates. Four informative single nucleotide polymorphisms (SNP) were identified and real-time PCR genotyping assays were designed and tested against additional diverse sets of bacterial isolates. Alleles of the first SNP, wbdR sp-11, were tested against 147 E. coli O157:H7, 33 E. coli O157:non-H7, and 123 non-E. coli O157 isolates. The wbdR sp-11 A allele had 100% sensitivity and specificity for E. coli O157:H7 isolates. Alleles of the second and third SNPs, per sp-11 and flhC sp-1, associate with sorbitol fermenting E. coli O157:NM (26/26, sensitivity 100%) or non-sorbitol fermenting E. coli O157:H7 isolates (1/315, specificity 99.7%; 3/315, specificity 99.1%, respectively). Alleles of the last SNP, tir sp-1, associate with human and bovine isolates, with 2% (2/98) of the human isolates and 35% (23/65) of bovine isolates having the tir-sp1 A allele (p< 0.000001). The combination of all four SNPs is a powerful tool for typing E. coli O157:H7 isolates and evaluating their potential to cause disease in humans.

Last Modified: 11/23/2014
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