|Chitko Mckown, Carol|
Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: February 27, 2004
Publication Date: May 1, 2004
Citation: Bono, J.L., Keen, J.E., Clawson, M.L., Heaton, M.P., Fox, J.M., Chitko McKown, C.G., Laegreid, W.W. 2004. Discrimination of Escherichia coli O157:H7 from other bacteria by genotyping single nucleotide polymorphisms [abstract]. American Society for Microbiology. Paper No. C-311. Technical Abstract: E. coli O157:H7 is the major cause of hemorrhagic colitis and hemolytic uremic syndrome in the United States. Rapid detection of E. coli O157:H7 is important so that infection control interventions can be instituted to minimize transmission, contamination, and disease by this pathogen. A number of genes have been targeted for E. coli O157:H7 diagnostic PCR, but many genes are not exclusively diagnostic for E. coli O157:H7 because other bacteria may have one or more of these genes. For example, several PCR targets have been designed against genes from the E. coli O157 O-antigen operon that had high sensitivity and specificity for E. coli O157. However, these same PCR targets were not able to distinguish between shiga toxin containing E. coli O157:H7 and non-shiga toxin containing E. coli O157:non-H7 isolates. We hypothesized the existence of genomic differences in the E. coli O157 O-antigen operon between the E. coli O157:H7 and non-E. coli O157:non-H7 that could be useful to design a PCR test specific for E. coli O157:H7. The per and wbdR genes from the E. coli O157 O-antigen synthesis operon were sequenced from 18 E. coli O157:H7 isolates and 8 non-E. coli O157:H7 isolates. A single nucleotide polymorphism (SNP) from both genes, per-sp11 and wbdR-sp13, defined two alleles with E. coli O157:H7 isolates belonging to a group with allele A and E. coli O157:non-H7 isolates belonging to a group with allele B. Real-time PCR genotyping assays were designed against the two SNPs and tested against 147 E. coli O157:H7, 33 E. coli O157:non-H7, and 123 non-E. coli O157 isolates. Two Graph-Receiver Operator Characteristic (TG-ROC) analysis of cycle threshold (CT) values from the 303 isolates indicated an optimal CT cutoff value of 32.0 for wbdR-sp13 and 35.6 for per-sp11. Using these cutoff values, the wbdR-sp13 assay had a 100% sensitivity and specificity, while the per-sp11 assay had 99% sensitivity and specificity for the detection of E. coli O157:H7. These results suggest that per-sp11 and wbdR-wp13 are excellent candidates for use as targets in real-time PCR assays to quickly detect E. coli O157:H7.