A novel enrichment-free, low-volume filtration and rapid lysis (ELR) method in combination with real-time PCR for detection of Shiga toxin-producing Escherichia coli (STEC) in water

Consequences of Shiga toxin-producing Escherichia coli (STEC) infection can range in severity from asymptomatic infection to haemolytic uraemic syndrome, renal failure and death. Groundwater-derived drinking water is an important route for STEC transmission. Detection of STEC in water is crucial for timely response and public health interventions; however, currently used culture-based methods are time-consuming and laborious. Therefore, there is a need for rapid methods that maintain high sensitivity and specificity [1]. We describe a novel, sensitive, enrichment-free water filtration method using a convenient sample volume (100 ml) to detect DNA markers of STEC serogroups and virulence factors within 6 h. Quantitative real-time PCR (qPCR) was used to detect and quantify the most common STEC infection-associated serogroups globally, O157 and O26. Real-time PCR was used to detect genetic determinants of STEC virulence ( stx1, stx2 and eae genes) and specific marker genes for the clinically relevant serogroups O111, O103, O145 and O104. Results showed that the novel method can detect as low as 5 c.f.u. ml -1 of STEC in water. The limit of detection for O157 and O26 qPCR assays was two and six copies, respectively. Groundwater and surface water samples ( n=28) were collected and processed using the novel method. STEC O157 and O26 serogroups were detected in 23 out of 28 (82.1%) samples (mean 5.2×10 4 copies/reaction) and 19 out of 28 (67.9%) samples (mean 7.83×10 4 copies/reaction), respectively. Shiga toxin genes stx1 or stx2 were detected in 15 out of 28 (53.6%) and 9 out of 28 (32.1%) samples, respectively. The virulence factor intimin gene eae was detected in 24 out of 28 (85.7%) samples. STEC serogroups O111, O103, O145 and O104 were detected in 15 out of 28 (53.6%), 10 out of 28 (35.7%), 11 out of 28 (39.3%) and 15 out of 28 (53.6%) samples, respectively. This novel method reproducibly detects low copies of STEC in low-volume fresh water and has the potential to be used for the detection and quantification of waterborne bacterial pathogens.