Occurrence of Viable E. Coli in Biofilm of a Drinking Water Supply System

The aim of any drinking water supplier is to bring to the consumer potable water of an identical quality to thai leaving the treatment plant. However, it has been well documented that water, which reaches the consumer's tap, is often of inferior microbiological quality, when compared to that which left the waterworks. Therefore the analytical systems used for monitoring of microflora in potable water must be quick to perform and able to detect the majoi groups of concern. Untreated water contains many bacteria, protozoa, fungi and viruses. Some of them may pass into drinking water and become established in biofilm, found on the inner surfaces of the drinking water distribution system. Attached organisms have been found to be generally active in absorbing nutrients as well as resistant to environmental stress such as starvation, heavy metals and chlorine and disinfection. Analyses of biofilm in drinking water are receiving increasing attention, because biofilm represents majority of biomass in the distribution network. It is quite convenient to detect bacteria in situ on the surface using FISH (fluorescence in situ hybridization) method. This method has become increasingly important for the drinking water industry and environmental microbiology because it is (i) quick, (ii) able to detect active but non-culturable cells (ABNC), which can still pose danger to the consumer but are not detectable by traditional methods, (iii) suitable for liquid samples as well as for solid, such as surfaces or biofilm, (iv) cheap enough to be used on a routine basis, and (v) has a potential for automatization. The ability to detect ABNC cells is particularly important as it has been shown that ABNC forms of bacteria are no longer capable of growing on conventional bacteriological media but conserve the pathogenic factors. The two most popular probe types are DNA probes and PNA probes. Peptide nucleic acid (PNA) molecules are DNA mimics in which the negatively charged sugar-phosphate backbone of DNA is replaced with a non-charged polyamide backbone. While being more expensive, PNA probes are superior to DNA probes because they produce higher signal intensity and require less stringent hybridization conditions. Moreover, using a PNA probe the time of hybridization can be reduced. As the PNA hybridization occurs in higher temperature, compared to DNA probes, unspecific binding is reduced. The challenge of bacteria detection by FISH on the surface is, however, to reduce the number of processing steps during the hybridization and thus to reduce the risk of cells loss. Another major problem with detection of bacteria in drinking water/biofilm is low signal to noise ratio. It is also preferable that the analysis protocol is as short and simple as possible. We have optimized the protocol in such way that the analysis time is even shorter while the obtained signal to noise ratio is higher and unspecific binding is kept to the minimum. Although both kinds of probes are able to detect ABN cells they, as such, do not provide information on how viable the cell is. For this other viability measurements, such as CTC staining in combination with FISH or methods, excluding the amplification of 16s RNA from dead cells (FISH-DVC, direct viable count), should be applied. In our earlier studies by using FISH-DVC method with PNA probes we showed that viable E. coli is present in biofilm in many water supply systems of Europe (Juhna et al 2007a) and that E. coli than can persist in biofilm for long time remaining in an unculturable form (Juhna et al 2007b). However this study did not show correlation off. coli number with any other parameters in water (source type, temperature, water treatment methods, biostability, water residence time, etc). The source of E. coli was also not identified. Here we present further study which aims to investigate whether viable E. coli can pass though water treatment plant and what is the role of biofil