Characterization of Shiga-toxin producing E.coli (STEC) and enteropathogenic E.coli (EPEC) using multiplex Real-Time PCR assays for stx(1), stx(2), eaeA

BACKGROUND AND OBJECTIVE: Diarrheal disease is still a major health problem, especially in developing countries, where it is considered as one of the leading causes of morbidity and mortality especially in children. Studies showed that Diarrheagenic E. coli (DEC) such as STES and EPEC strains are among the most prevalent causative agents in acute diarrhea, particularly in children. Aim of the present study was to investigate the presence and the frequency of STEC and EPEC as etiologic agent of diarrhea in children less than 2 years of age with diarrhea in Shiraz. MATERIALS AND METHODS: A total of 285 stool samples were collected from patients with diarrhea in Shiraz, in 2012. Diarrheagenic E. coli (DEC) strains were isolated by standard biochemical analysis. In this study, we used multiplex Real time PCR and single PCR to detect the presence of indicator genes stx(1), stx(2) and eaeA for STEC and EPEC strains, respectively. RESULTS: A total of 285 stool samples were tested in which 49 (17%) were identified as contaminated with E. coli by biochemical tests. Out of total samples, 15 STEC (31%) and 13 EPEC (27%) were identified using multiplex Real-Time PCR assay. Among STEC isolates, 2 strains were stx(1)(+), 8 isolates stx(2)(+), 3 isolates were stx(1)(+), stx(2)(+) and 2 isolates were stx(1)(+), stx(2)(+), eaeA(+). CONCLUSION: In this study, we found rather high occurrence of STEC and EPEC virulence genes in children with diarrhea. The results of this study showed that, real time PCR can be used as a replacement for conventional PCR assay in the detecting virulence genes of STEC and EPEC strains. Real-time PCR offers the advantage of being a faster, more robust assay, because it does not require post-PCR procedures to detect amplification products.