Detection of Escherichia coli in biofilms from pipe samples and coupons in drinking water distribution networks

Juhna, T., Birzniece, D., Larsson, S., Zulenkovs, D., Sharipo, A., Azevedo, N.F., Menard-Szczebara, F., Castagnet, S., Feliers, C. and Keevil, C.W. (2007) Detection of Escherichia coli in biofilms from pipe samples and coupons in drinking water distribution networks. Applied and Environmental Microbiology, 73, 7456-7464. (doi:10.1128/AEM.00845-07).

Abstract

Fluorescence in situ hybridization (FISH) was applied for direct detection of Escherichia coli on pipe surfaces and coupons in drinking water distribution networks. Old cast iron main pipes were removed from water distribution networks in France, England, Portugal and Latvia and E. coli was analyzed in the biofilm. In addition, 44 flat coupons made of cast iron, PVC or stainless steel were placed into and continuously exposed to water on 15 locations of 6 distribution networks in France and Latvia and examined after one to six months exposure to the drinking water. In order to increase signal intensity a peptide nucleic acid (PNA) 15-mer probe was used in the FISH screening for the presence or absence of E. coli on the surface of pipes and coupons, thus reducing occasional problems of autofluorescence and low fluorescence of the labeled bacteria. For comparison, cells were removed from the surfaces and examined with culture-based or enzymatic (detection of -D-glucuronidase) methods. An additional verification was made using PCR. Culture method indicated presence of E.coli in 1 out of 5 pipes whereas all pipes were positive with FISH methods. E.coli were detected in 56% of coupons using PNA FISH but no E.coli were detected using culture or enzymatic methods. PCR analyses confirmed the presence of E. coli in samples which were negative according to the culture-based and enzymatic methods. The viability of E. coli cells in the samples was demonstrated by the cell elongation after resuscitation in low nutrient medium supplemented with pipemidic acid, suggesting that the cells were present in an active but non-culturable state (ABNC), unable to grow on agar media. E. coli contributed to approximately 0.001 - 0.1 % of the total bacterial number in the samples. The presence and number of E. coli did not correlate with any of physical and/or chemical characteristic of the drinking water (e.g. temperature, chlorine or biodegradable organic matter concentration).
The study showed that E. coli is present in the biofilms of drinking water networks in Europe. Some of the cells are metabolically active but are often not detected due to limitations of traditionally used culture-based methods, indicating that biofilm should be considered as a reservoir that must be investigated further in order to evaluate the risk for human health.

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Contributors

Author: C.W. Keevil

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