Molecular analysis of the bacterial drinking water community with respect to live/dead status

The assessment of the physiological state of the bacteria in drinking water is a critical issue, especially with respect to the presence of pathogenic bacteria. Though molecular methods can provide insight into the taxonomic composition of the drinking water microflora, the question if a bacterial species is alive or dead still needs to be addressed. To distinguish live and dead bacteria at the taxonomic level, we combined three methods: i) a staining procedure indicating membrane-injured cells (using SYTO9 and Propidium Iodide) that is considered to distinguish between live and dead cells, ii) Fluorescence Activated Cell Sorting (FACS) of the membrane injured and intact bacteria, and iii) molecular analyses of the RNA extracted from the bacteria before and after sorting to analyse the bacterial community at the species level. By staining and FACS analysis the drinking water bacteria could be separated according to their different membrane integrities, and RNA could be extracted from the live and dead sorted bacterial fractions. 16S rRNA based fingerprints revealed a diverse bacterial community in the drinking water samples with the majority being represented by 31 identified phylotypes. Most of the phylotypes referenced belonged to the phyla Proteobacteria (Alpha-, Beta-, Gamma-), Cyanobacteria and Bacteroidetes, and were mostly related to freshwater bacteria. 90% of the total phylotypes could be recovered after FAC-Sorting; 32% of the phylotypes occurred only in the "live" sorted fraction, 21% only in the "dead" sorted fraction, and 46% occurred in both fractions.