Y-021. Detection and Characterization of eae Variants Found in STEC Positive Stool Samples

P. A. Michel, J. Kase;
North Carolina State Lab of Pub. Hlth., Raleigh, NC.

Background: Enteropathogenic E coli (EPEC) and shiga toxin-producing E coli (STEC) are categories of diarrheagenic E. coli that cause attaching and effacing (A/E) lesions in the intestinal epithelial cells of the infected hosts. The outer membrane protein, intimin, encoded by the eae gene has been shown to be a genetic determinant for the production of A/E lesions and is also used as a molecular marker for identification of EPEC infections. Furthermore, there have been at least 25 genetic variants of the eae gene isolated from humans and animals to date. The North Carolina State Lab of Public Health (NCSLPH) is currently investigating the prevalence and specific variant form of the eae gene present in human stool broth samples submitted to the lab that have previously been tested positive for STEC. Methods: Using a real-time PCR method, all STEC positive stool broth samples submitted to the NCSLPH from January 2006 to the present were tested for the presence of the eae gene. To discriminate between eae gene variants (eaeβ1, eaeγ1, eaeε1, eaeθ/γ2, eaeξR/β2B, and eaeζ ) a PCR strategy was employed that utilized primers specific to each eae genetic variant. Results: Of 81 STEC positive samples tested at the NCSLPH, 79(97.5%) were eae positive by real-time PCR. Conventional PCR showed that of the 79 eae positives, 30(38%) were eaeε1, 15(19%) eaeβ1, 11(14%) eaeγ1, 9(11%) eaeξR/β2B, and 14(18%) that were not determined. None of the samples tested contained the eaeζ variant. Conclusions: The initial results of this study have shown that nearly all of the STEC positive samples submitted to the NCSLPH also contained the eae gene. Moreover, eaeε1 is the most common eae gene variant associated with STEC positive stool samples. The 14 eae positive samples that were unable to be further sub-typed by PCR could potentially be variants yet to be screened by PCR or novel eae variants. To address these assumptions, PCR is being carried out using a larger variety of eae specific primers. In the case of inconclusive or ambiguous results from this PCR method, direct nucleotide sequencing of eae PCR fragments will be carried out.