Isolation of new bacterial species from drinking water biofilms and proof of their in situ dominance with highly specific 16S rRNA probes

Abstract

A polyphasic approach involving cultivation, direct viable counts, rRNA-based phylogenetic classification, and in situ probing was applied for the characterization of the dominant microbial population in a municipal drinking water distribution system. A total of 234 bacterial strains cultivated on R2A medium were screened for bacteria affiliated with the in situ dominating beta subclass of Proteobacteria. The isolates were grouped according to common features of their cell and colony morphologies, and eight representative strains were used for 16S rRNA sequencing and the development of a suite of strain-specific oligonucleotide probes. Phylogenetic analysis indicated that all of the isolates were hitherto unknown bacteria. Three of them, strains B4, B6, and B8, formed a separate cluster of closely related organisms within the beta1 subclass of Proteobacteria. In situ probing revealed that (i) 67 to 72% of total bacteria, corresponding to more than 80% of beta-subclass 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 population could be shown to be in a viable but nonculturable (VBNC) state by using a direct viable count approach. The comparison of isolation frequencies with the in situ abundances of the eight investigated strains revealed differences in their culturability, indicating variable ratios of culturable to VBNC cells among the strains. The further characterization of biofilms throughout the distribution network demonstrated strains B6 and B8 to be dominant bacterial strains in groundwater and distribution system biofilms. The other strains could be found at various frequencies in the different parts of the distribution system; several strains appeared exclusively in drinking water biofilms obtained from a house installation system.

Bacterial activity and community composition in stream water and biofilm from an urban river determined by fluorescent in situ hybridization and DGGE analysis

“…During the di¡erent months sampled, bacteria on polycarbonate slides were dominated by the L subclass of Proteobacteria. This group of bacteria has been described as the most morphologically diverse group within bio¢lms [5], and a dominant group in fresh water systems such as oligotrophic lakes, drinking water bio¢lms [29,36,37], river snow [26] and eutrophic rivers [9,30]. It seems to be a common feature of most aquatic environments.…”

Evaluation of cell activity and of methods for the cultivation of bacteria from a natural lake community

“…A typical medium used for e¡ective cultivation of bacteria from aquatic environments is R2A [23]. This complex medium has been used successfully for growth of a high fraction of bacterial cells in seawater or wastewater [8], and for the isolation the dominating bacterial strains from municipal drinking water [21]. In our synthetic freshwater medium MPN counts were higher than in R2A medium.…”

“…Cells which stop growing after a few divisions may be classi¢ed as viable but nonculturable (VBNC, [37]). In drinking water, up to 65% of the bacteria appeared to be in this state [21].…”

Detection of Legionella spp. by fluorescent in situ hybridization in dental unit waterlines

“…It is thus essential to use a more powerful technique for legionellae detection in environmental samples. Fluorescent in situ hybridization (FISH) using genus-specific fluorescently labelled oligonucleotide probe, complementary to a conserved target sequence of the 16S rRNA region, has been shown to be a useful method for the detection and identification of slow growing, fastidious or nonculturable micro-organisms without cultivation (Amann et al 1991;Kalmbach et al 1997;Declerck et al 2003). Two problems were encountered by the use of FISH for detecting bacteria in natural water: (i) the low level of targeted rRNA (low fluorescence) and (ii) the inability to distinguish viable from nonviable cells (Villarino et al 2000;Garcia-Armisen and Servais 2004).…”