A novel Enrichment-free, Low-volume filtration and Rapid lysis method (ELR) 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 (HUS), 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. 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 hours. Quantitative real-time Polymerase Chain Reaction (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 CFU/mL of STEC in water. The limit of detection for O157 and O26 qPCR assays was 2 and 6 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 23/28 (82.1%) samples (mean 5.2x104 copies/reaction) and 19/28 (67.9%) samples (mean 7.83x104 copies/reaction), respectively. Shiga toxin genes stx1 or stx2 were detected in 15/28 (53.6%) and 9/28 (32.1%) samples, respectively. Virulence factor intimin gene eae was detected in 24/28 (85.7%) samples. STEC serogroups O111, O103, O145 and O104 were detected in 15/28 (53.6%), 10/28 (35.7%), 11/28 (39.3%) and 15/28 (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 detection and quantification of waterborne bacterial pathogens.