Impact of pollution and seasonal changes on microbial community structure in surface water.

We studied the differences in a microbial community structure with respect to the water pollution level and seasonal changes. The determination of phylogenetic groups of Bacteria and Archaea was done using fluorescent in situ hybridization (FISH). The total number of microorganisms was determined by direct counting of DAPI (4',6-diamidino-2-phenylindole) stained samples using a fluorescence microscope. Our results showed that the microbial community structure was significantly dependent on the level of water pollution, both in absolute microbial counts and in relative abundance of phylogenetic groups. For surface water with anthropogenic pollution, the microbial community with significant proportion of Betaproteobacteria and Cytophaga-Flavobacterium was characteristic. Gammaproteobacteria were significant in municipal waste water. In microbial communities with low numbers of microorganisms (e.g. non-polluted water and some industrial waste water) represented the significant component groups Alphaproteobacteria and Archaea. The impact of seasonal changes on the microbial distribution was not significant.

Evaluation of methods for isolation of DNA for polymerase chain reaction (PCR)-based identification of pathogenic bacteria from pure cultures and water samples.

Polymerase chain reaction (PCR) provides a reliable detection of pathogenic bacteria in water samples. However, this method can be adversely influenced by the purity of the DNA template. This is a particularly important obstacle when the bacterial DNA is directly extracted from water samples. In this study we compared the suitability of 8 different methods for isolation of bacterial DNA from pure cultures and 10 different methods for isolation of DNA from water samples. The quality of extracted DNA was assessed by PCR amplification of target sequences derived from uid (E. coli and Shigella sp.), tuf (Enterococcus sp.) and hns (Salmonella sp.). Results indicated that there are differences among the methods tested and only a few of them gave satisfactory results. The method based on alkaline lysis of bacterial suspension, which was developed in our laboratory, seemed to be efficient enough for the detection of bacteria from pure cultures. Detection of bacteria directly from water samples was more difficult. The modified method developed by Slusarenko was found as the best of the tested methods.

Specific detection of Escherichia coli isolated from water samples using polymerase chain reaction targeting four genes: cytochrome bd complex, lactose permease, beta-D-glucuronidase, and beta-D-galactosidase.

AIMS: To develop a PCR-based method for reliable detection of Escherichia coli that enables its differentiation from biochemically and phylogenetically related bacteria. METHODS AND RESULTS: Using multiplex PCR targeting four genes (cytochrome bd complex, lactose permease, beta-d-glucuronidase, and beta-d-galactosidase) the possibility of specific detection of various control E. coli strains was tested. It was found that four PCR fragments of the predicted size were observed only for E. coli strains, but not for relatives as close as Shigella sp. or other enterobacteria. Not surprisingly, this method enabled us to identify also E. coli strains which did not exhibit the beta-d-glucuronidase activity. Our multiplex PCR was also successfully used for identification of 95 environmental isolates of E. coli. CONCLUSIONS: The developed PCR-based method, in which four genes coding for lactose permease, cytochrome bd complex, beta-d-glucuronidase, and beta-d-galactosidase, serve as target DNA sequences, allows precise and reliable detection of E. coli strains. SIGNIFICANCE AND IMPACT OF THE STUDY: The suggested approach increases the specificity of detection of E. coli since it enables to distinguish E. coli from Shigella sp. and other relative enterobacteria.

Direct detection of bacterial faecal indicators in water samples using PCR.

The presence of enteric pathogens in water resources represents a serious risk for public health. Therefore, their precise detection, and especially detection of E. coli, which is obviously regarded as the main indicator of faecal contamination of water, is an essential step in ensuring bacterial safety of water. Numerous PCR protocols for detection of E. coli have been published to date. They are usually based on amplification of regions derived from lacZ (beta-D-galactosidase) and uidA (beta-D-glucuronidase) gene sequences. However, these methods are not universal enough for precise detection of all E. coli strains found in water samples. We developed a novel triplex PCR method for detection of E. coli in which cyd gene coding for cytochrome bd complex was co-amplified along with lacZ and uidA genes. Our triplex PCR approach significantly increases the specificity and reliability of E. coli detection in water samples. This approach allowed us to distinguish Shigella flexneri from E. coli. In addition, we were able to detect even non-coliform Klebsiella and Raoutella spp., some of which can also cause infections to humans.