Characterization of enteroaggregative Escherichia coli (EAEC) clinical isolates and their antibiotic resistance pattern

Abstract

Objectives: Enteroaggregative Escherichia coli (EAEC) strains are an emerging type of diarrheagenic E. coli. The aim of this study was to investigate the frequency of EAEC in children with diarrhea by polymerase chain reaction (PCR) method targeting the pCVD432 gene. The presence of virulence genes including aggR, aggA, aafA, aap, and astA was also investigated by PCR, for the differentiation of typical and atypical EAEC strains. We also sought to determine the antibiotic resistance pattern of the isolated strains.

Methods: Stool samples were collected from 140 children with diarrhea at Besat Hospital, Hamadan, Iran, from July 2007 to May 2008. The specimens were cultured for E. coli, which was identified using standard methods. E. coli strains were screened for EAEC by PCR and HeLa cell line adherence methods. For each sample, five single colonies (700 E. coli strains) from original MacConkey plates were examined for pCVD432, aggR, aggA, aafA, aap, and astA genes using PCR. The EAEC adherence patterns were examined by HeLa cell adherence method. Antimicrobial susceptibility testing was performed as the Clinical and Laboratory Standards Institute (CLSI) guidelines.

Results: Overall, 15 (10.7%) EAEC strains were identified in 140 diarrhea cases by PCR. Out of these isolates, EAEC were detected in 13 (86.7%) by the HeLa cell assay. The aggR regulon was present in 11 (73.3%) strains. Several different combinations of the virulence markers were found among the EAEC isolates. The most prevalent (20%) combination was aggR-aap-astA. The EAEC isolates exhibited resistance to ampicillin (100%), erythromycin (100%), cephalothin (78.6%), co-trimoxazole (71.4%), tetracycline (64.3%), and nalidixic acid (57.1%) and reduced resistance to ciprofloxacin (42.9%) and norfloxacin (7.1%).

Conclusions: EAEC is a diarrheal pathogen of emerging importance. Correlation between pCVD432 PCR and the HeLa cell line assay was confirmed in children with diarrhea. In comparison to the assay for aggregative adherence, the EAEC PCR has been found to be simple and specific in many epidemiological studies. The typical EAEC (73.3%) strains (with pCVD432 and aggR genes) identified in this study were heterogeneous with respect to virulence genes. This study also showed that EAEC isolates were highly resistant to tetracycline, co-trimoxazole, and ampicillin, which are the most commonly used antibiotics in our area.