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

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Abstract

Escherichia coli O157:H7 and Salmonella are pathogenic microorganisms that can cause severe gastrointestinal illness in humans. These pathogens may be transmitted in a variety of ways, including food and water. The presence of Salmonella and E. coli O157:H7 in surface waters constitutes a potential threat to human health when used for either drinking or recreation. As with most waterborne pathogens, Salmonella and E. coli O157:H7 are difficult to detect and enumerate with accuracy in surface waters due to methodological limitations. The aim of this study was to develop a protocol for the detection of Salmonella spp., E. coli O157:H7 and E. coli virulence genes (stx1, stx2 and eae) in water using a single enrichment step and PCR. In spiked water samples, PCR results showed high sensitivity (<3 CFU/L) for both microorganisms. The protocol developed in this study has been applied in different surface waters in association with microbiological and physical analysis. The frequency of PCR positive samples was 33% for Salmonella and 2% for E. coli O157:H7 producing intimin (eae) and Shiga-like toxin I (stx1). Moreover, the finding of amplicons corresponding to eae and stx1 genes in the absence of E. coli O157:H7 suggested the possible presence of other pathogenic bacteria that carry these genes (e.g. EHEC, Shigella strains). The results obtained showed that the developed protocol could be applied as a routine analysis of surface water for the evaluation of microbiological risks.

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References

  • Ahmed, W., Sawant, S., Huygens, F., Goonetilleke, A., & Gardner, T. (2009). Prevalence and occurrence of zoonotic bacterial pathogens in surface waters determined by quantitative PCR. Water Research, 43, 4918–4928.

    Article  CAS  Google Scholar 

  • Avery, L. M., Williams, A. P., Killham, K., & Jones, D. L. (2008). Survival of Escherichia coli O157:H7 in waters from lakes, rivers, puddles and animal-drinking troughs. Science of the Total Environment, 389, 378–385.

    Article  CAS  Google Scholar 

  • Bertrand, R., & Roig, B. (2007). Evaluation of enrichment-free PCR-based detection on the rfbE gene of Escherichia coli O157—Application to municipal wastewater. Water Research, 41, 1280–1286.

    Article  CAS  Google Scholar 

  • Collazo, C. M., & Galan, J. E. (1997). The invasion-associated type-III protein secretion system in Salmonella – a review. Gene, 192, 51–59.

    Article  CAS  Google Scholar 

  • Dharmasiri, U., Witek, M. A., Adams, A. A., Osiri, J. K., Hupert, M. L., Bianchi, T. S., et al. (2010). Enrichment and detection of Escherichia coli O157:H7 from water samples using an antibody modified microfluidic chip. Analytical Chemistry, 82, 2844–2849.

    Article  CAS  Google Scholar 

  • Dorner, S. M., Anderson, W. B., Gaulin, T., Candon, H. L., Slawson, R. M., Payment, P., et al. (2007). Pathogen and indicator variability in a heavily impacted watershed. Journal of Water and Health, 05.2, 241–257.

    Google Scholar 

  • Duris, J. W., Haack, S. K., & Fogarty, L. R. (2009). Gene and antigen markers of Shiga-toxin producing E.coli from Michigan and Indiana river water: occurrence and relation to recreation al water quality criteria. Journal of Environmental Quality, 38, 1878–1886.

    Article  CAS  Google Scholar 

  • EFSA (2010). The community summary report on trends and sources of zoonoses and zoonotic agents in the European Union in 2008. The EFSA Journal, 1496.

  • Feder, I., Nietfeld, J. C., Galland, J., Yeary, T., Sargent, J. M., Oberst, R., et al. (2001). Comparison of cultivation and PCR-hybridization for detection of Salmonella in porcine fecal and water samples. Journal of Clinical Microbiology, 39, 2477–2484.

    Article  CAS  Google Scholar 

  • Hrudey, S. E., & Hrudey, E. J. (2004). Safe drinking water: Lessons from recent outbreaks in affluent nations. London: IWA.

    Google Scholar 

  • Hrudey, S. E., Payment, P., Huck, P. M., Gillham, R. W., & Hrudey, E. J. (2003). A fatal waterborne disease epidemic in Walkerton, Ontario: Comparison with other waterborne outbreak in the developed world. Water Science and Technology, 47, 7–14.

    CAS  Google Scholar 

  • Hu, Y., Zhang, Q., & Meitzer, J. C. (1999). Rapid and sensitive detection of Escherichia coli O157:H7 in bovine faeces by a multiplex PCR. Journal of Applied Microbiology, 87, 867–876.

    Article  CAS  Google Scholar 

  • ISS (2007a). Metodi Analitici di riferimento per le acque destinate al consumo umano ai sensi del DL.vo 31/2001. Metodi Microbiologici. Edited by: L. Bonadonna, M. Ottaviani. Rapporti ISTISAN, 07/5, Istituto Superiore di Sanità.

  • ISS (2007b). Metodi Analitici di riferimento per le acque destinate al consumo umano ai sensi del DL.vo 31/2001. Metodi Chimici. Edited by: L. Bonadonna, M. Ottaviani. Rapporti ISTISAN, 07/31, Istituto Superiore di Sanità.

  • James, C. E., Stanley, K. N., Allison, H. E., Flint, H. J., Stewart, C. S., Sharp, R. J., et al. (2001). Lytic and lysogenic infection of diverse Escherichia coli and Shigella strains with a verocytotoxigenic bacteriophage. Applied and Environmental Microbiology, 67, 4335–4337.

    Article  CAS  Google Scholar 

  • Kuhnert, P., Boerlin, P., & Frey, J. (2000). Target genes for virulence assessment of E. coli isolates from water, food and the environment. FEMS Microbiology Reviews, 24, 107–117.

    Article  CAS  Google Scholar 

  • Lemarchand, K., & Lebaron, P. (2003). Occurrence of Salmonella spp. and Cryptosporidium spp. in French coastal watershed: relationship with fecal indicators. FEMS Microbiology Letters, 218, 203–209.

    Article  CAS  Google Scholar 

  • Liu, Y., Wang, C., Tyrrell, G., & Li, X. F. (2010). Production of Shiga-like toxins in viable but non culturable Escherichia coli O157:H7. Water Research, 44, 711–718.

    Article  CAS  Google Scholar 

  • Malorny, B., Hoofar, J., Bunge, C., & Helmuth, R. (2003). Multicenter validation of the analytical accuracy of Salmonella PCR: towards and international standard. Applied and Environmental Microbiology, 69, 290–296.

    Article  CAS  Google Scholar 

  • Marucci, P. L., Olivera, N. L., Brugnoni, L. I., Sica, M. G., & Cubitto, M. A. (2010). The occurrence of Shiga toxin-producing Escherichia coli in bathing water of the Sierra de la Ventana region, Buenos Aires Province, Argentina. Environmental Monitoring and Assessment. doi:10.1007/s10661-010-1488-2.

    Google Scholar 

  • Moganedi, K. L. M., Goyvaerts, E. M. A., Venter, S. N., & Sibara, M. M. (2007). Optimization of the PCR-invA primers for the detection of Salmonella in drinking and surface waters following a pre-cultivation step. Water SA, 33, 195–202.

    CAS  Google Scholar 

  • Mull, B., & Hill, V. R. (2009). Recovery and detection of Escherichia coli O157:H7 in surface water, using ultrafiltration and Real-Time PCR. Applied and Environmental Microbiology, 75, 3593–3597.

    Article  CAS  Google Scholar 

  • Nwachuku, N., & Gerba, C. P. (2008). Occurence and persistence of Escherichia coli O157:H7 in water. Reviews in Environmental Science and Biotechnology, 7, 267–273.

    Article  CAS  Google Scholar 

  • Shelton, D. R., Karns, J. S., Higgins, J. A., Van Kessel, J. A., Perdue, M. L., Belt, K. T., et al. (2006). Inpact of microbial diversity on rapid detection of enterohemorragic Escherichia coli in surface waters. FEMS Microbiology Letters, 261, 95–101.

    Article  CAS  Google Scholar 

  • Strauch, E., Hammerl, J. A., Konietzny, A., Schneiker-Bekel, S., Arnold, W., Goesmann, A., et al. (2008). Bacteriophage 2851 is a prototype phage for dissemination of the Shiga toxin variant gene 2c in Escherichia coli O157:H7. Infection and Immunity, 76, 5466–5477.

    Article  CAS  Google Scholar 

  • Sugumar, G., Chrisolite, B., Velayutham, P., Selvan, A., & Ramesh, U. (2008). Occurence and season variation of bacterial indicators of faecal pollution along Thoothukudi Coast, Tamil Nadu. Journal of Environmental Biology, 29, 387–391.

    CAS  Google Scholar 

  • Thompson, D. E., Rajal, V. B., De Batz, S., & Wuertz, S. (2006). Detection of Salmonella spp. in water using magnetic capture hybridization combined with PCR or Real-Time PCR. Journal of Water and Health, 04.1, 67–75.

    Google Scholar 

  • Touron, A., Berthe, T., Pawlak, B., & Petit, F. (2005). Detection of Salmonella in environmental water and sediment by a nested-multiplex polymerase chain reaction assay. Research in Microbiology, 156, 541–553.

    Article  CAS  Google Scholar 

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Correspondence to Elisabetta Carraro.

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Bonetta, S., Borelli, E., Bonetta, S. et al. Development of a PCR protocol for the detection of Escherichia coli O157:H7 and Salmonella spp. in surface water. Environ Monit Assess 177, 493–503 (2011). https://doi.org/10.1007/s10661-010-1650-x

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  • DOI: https://doi.org/10.1007/s10661-010-1650-x

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