Recovery and Detection of Escherichia coli O157:H7 in Surface Water, Using Ultrafiltration and Real-Time PCR

Abstract

Enterohemorrhagic Escherichia coli O157:H7 (EHEC O157:H7) outbreaks have revealed the need for improved analytical techniques for environmental samples. Ultrafiltration (UF) is increasingly recognized as an effective procedure for concentrating and recovering microbes from large volumes of water and treated wastewater. This study describes the application of hollow-fiber UF as the primary step for concentrating EHEC O157:H7 seeded into 40-liter samples of surface water, followed by an established culture/immunomagnetic-separation (IMS) method and a suite of real-time PCR assays. Three TaqMan assays were used to detect the stx1, stx2, and rfbE gene targets. The results from this study indicate that approximately 50 EHEC O157:H7 cells can be consistently recovered from a 40-liter surface water sample and detected by culture and real-time PCR. Centrifugation was investigated and shown to be a viable alternative to membrane filtration in the secondary culture/IMS step when water quality limits the volume of water that can be processed by a filter. Using multiple PCR assay sets to detect rfbE, stx1, and stx2 genes allowed for specific detection of EHEC O157:H7 from strains that do not possess all three genes. The reported sample collection and analysis procedure should be a sensitive and effective tool for detecting EHEC O157:H7 in response to outbreaks of disease associated with contaminated water.

Tangential-Flow Ultrafiltration with Integrated Inhibition Detection for Recovery of Surrogates and Human Pathogens from Large-Volume Source Water and Finished Drinking Water

“…These calculations were done to demonstrate the estimated original sample volume that was analyzed during molecular analyses. These back calculations have often not been reported in previously published studies involving concentration from large volumes of water combined with realtime PCR (18,30,34). Not unexpectedly (due to greater turbidity) nearly a 10-fold difference in the original sample volumes analyzed was found between SW and DW.…”

Improving Salmonella determination in Sinaloa rivers with ultrafiltration and most probable number methods

“…Several authors emphasize the benefits of the use of UFS to recover waterborne microorganisms due to its properties that include: (1) removal of low molecular weight inhibitors, (2) reduction of membrane fouling, and (3) filtration of large volumes of water without pH modification, which protects microorganisms from stressful conditions and facilitates recovery (Leskinen and Lim 2008;Mull and Hill 2009;Grassi et al 2010). However, most studies have determined the recovery efficiencies of UFS variants (Winona et al 2001;Hill et al 2005;Olszewski et al 2005) without examining the impact of UFS implementation on the determination of pathogens (Hernandez-Morga et al 2009).…”

Development of a PCR protocol for the detection of Escherichia coli O157:H7 and Salmonella spp. in surface water

“…In order to achieve successful detection in water samples, we have proposed a protocol based on a single enrichment step and the PCR. In recent years, the development of molecular methods for the amplification of specific gene sequences has produced rapid and sensitive techniques (PCR, PCR-ELISA, RT-PCR or real-time PCR) for detecting pathogens in waters (Ahmed et al 2009;Liu et al 2010;Mull and Hill 2009;Touron et al 2005). However, considering the low concentration of pathogens in water, an enrichment step is frequently required to improve the sensitivity.…”