Detection of enterococci with rRNA targeted DNA probes and their use for hygienic drinking water control

Abstract

Enterococci are useful indicators of faecal contamination with their high abundance in faeces and long survival in the environment and the possibility of indicating the source of contamination by species identification has lead to discussion of whether enterococci would be more reliable faecal indicators than E. coli. In an attempt to facilitate rapid and accurate identification of enterococci, l6S rRNA targeted oligonucleotide probes were designed by computer-aided analysis of more than 4,000 rRNA sequences. Probes were labelled non-isotopically with digoxigenin and fluorescent dyes. Conditions for specific hybridisation were optimised for dot blot hybridisation and whole cell hybridisation for all probes. With a combination of two probes, all hygienically important enterococci could be detected and 24 biochemically identified environmental isolates also hybridised with one of these probes. A quantitative detection method with a high sensitivity was developed based on filtration of water samples through polycarbonate filters, a short incubation on agar and microcolony filter hybridisation with fluorescently labelled probes followed by epifluorescence microscopy. Within 8–20h sampling a specific identification of enterococcal microcolonies was possible. With this method 15/32 well- and tap-water sources from the Mains area were identified as being of substandard quality. The proposed method detects faecal contamination significantly earlier than conventional methods and could be helpful in the hygienic monitoring of drinking water.