N-208. Identification of Active Bacterial Communities in Drinking Water Using 16S rRNA-Based Sequence Analyses

R. P. Revetta, A. Pemberton, J. W. Santo Domingo;
US EPA, Cincinnati, OH.

DNA-based methods have considerably increased our understanding of the bacterial diversity of water distribution systems (WDS). However, as DNA may persist after cell death, the use of DNA-based methods cannot be used to describe metabolically-active microbes. In contrast, intracellular RNA is rapidly degraded in stressed cells and is unstable outside of the cell. Consequently, molecular surveys targeting 16S rRNA are better suited at identifying active bacterial communities in drinking water. In this study, we used hollow-fiber ultrafiltration to concentrate bacterial communities from 40 liters of tap water collected at different times from a single point-of-use. Total RNA was extracted from the microbial concentrates and then used to develop 16S rRNA-based clone libraries. Sixty-five operational taxonomic units (OTUs; at 98% identity or greater) were formed from 2,827 sequences. Phylogenetic analyses revealed that uncultured bacterial clones (UBC) were the most predominant group, representing 57% of OTUs, and 91% of total sequences in the samples analyzed. Within the UBC group, 44% of sequences were closely related to sequences retrieved from previous drinking water studies, including sequences retrieved from DNA-, RNA-, and culture-based studies. Overall, the results suggest that these bacteria are indeed among the most common metabolically active drinking water bacteria. Additionally, 8% of the UBC sequences showed 97% or less identity with sequences present in public databases. Other bacterial groups represented in this study included Proteobacteria (3% of total sequences analyzed), and cyanobacteria, actinobacteria, Firmicutes, Bacteroidetes, and Planctomycetes (less than 1% each). The results from this study further improve our understanding of the molecular diversity and bacterial population dynamics of WDS microbial communities. Moreover, these results provide the sequence foundation for the development of molecular assays that target active drinking water bacteria.