Abstract
Shiga-toxigenic Escherichia coli (STEC) and enteropathogenic E. coli (EPEC) are recognised to be significant contributors to intestinal and extra-intestinal disease and infantile diarrhoea worldwide. In New Zealand, STEC serotype O157:H7 is a notifiable pathogen. The routine use of sorbitol MacConkey (SMAC)-based agar to detect O157:H7 fails to detect many potentially pathogenic non-O157 E. coli serogroups and EPEC is not currently under surveillance. As a result, the contribution of these pathogens to diarrhoeal disease is likely to be underrepresented in governmental surveillance reports. The intent of this project was to develop a multiplex qRT-PCR TaqMan assay suitable for the detection of STEC (stx1 and stx2) and EPEC (eaeA/intimin) marker genes, and to use this assay to survey diarrhoeic stool samples sourced from Dunedin Hospital. Prevalences of 1.53% and 4.41% (95% CI) were found for STEC and EPEC respectively, with 75% of stx+ samples possessing STEC belonging to serogroups other than O157. A trend towards an increased rate of STEC infection amongst samples also tested for Helicobacter pylori was also noted, indicating possible confusion of symptoms by clinicians. The project also tested a number of DNA extraction methods and pre-enrichments to further adapt the assay to high-throughput diagnostic practice. The inexpensive Chelex-100 resin performed satisfactorily when compared with phenol-chloroform and PrepMan Ultra methods. Trypticase soy broth was found to offer the most consistent pre-enrichment for STEC compared to other commonly used non-selective liquid culture media. A 3 hour pre-enrichment of spiked faecal samples was found to boost the sensitivity of the qRT-PCR method to detect 2 x 10^2 CFU per g of faeces.