ISOLATION OF NEW BACTERIAL SPECIES FROM DRINKING-WATER BIOFILMS AND PROOF OF THEIR IN-SITU DOMINANCE WITH HIGHLY SPECIFIC 16S RIBOSOMAL-RNAPROBES

A polyphasic approach involving cultivation, direct viable counts, rRN A-based phylogenetic classification, and in situ probing was applied f or the characterization of the dominant microbial population in a muni cipal drinking water distribution system, A total of 234 bacterial str ains cultivated on R2A medium were screened for bacteria affiliated wi th the in situ dominating beta subclass of Proteobacteria. The isolate s were grouped according to common features of their cell and colony m orphologies, and eight representative strains were used for 16S rRNA. sequencing and the development of a suite of strain-specific oligonucl eotide probes, Phylogenetic analysis indicated that all of the isolate s were hitherto unknown bacteria, Three of them, strains B4, B6, and B 8, formed a separate cluster of closely related organisms within the b eta1 subclass of Proteobacteria. In situ probing revealed that (i) 67 to 72% of total bacteria, corresponding to more than 80% of beta subcl ass bacteria, could be encompassed with the strain-specific probes and (ii) the dominating bacterial species were culturable on R2A medium, Additionally, two-thirds of the autochthonous drinking water populatio n could be shown to be in a viable but nonculturable (VBNC) state by u sing a direct viable count approach, The comparison of isolation frequ encies with the in situ abundances of the eight investigated strains r evealed differences in their culturability, indicating variable ratios of culturable to VBNC cells among the strains, The further characteri zation of biofilms throughout the distribution network demonstrated st rains B6 and B8 to be dominant bacterial strains in groundwater and di stribution system biofilms, The other strains could be found at variou s frequencies in the different parts of the distribution system; sever al strains appeared exclusively in drinking water biofilms obtained fr om a house installation system.