Free-living protozoa in two unchlorinated drinking water supplies identified by phylogenic analysis of 18S rRNA gene sequences

Free-living protozoan communities in water supplies may include hosts for Legionella pneumophila and other undesired bacteria and also pathogens. This study aimed at identifying free-living protozoa in two unchlorinated groundwater supplies using cultivation-independent molecular approaches. For this purpose, samples (T <20 degrees C) of treated water, distributed water and distribution system biofilms were collected from supply A with a low concentration of natural organic matter (NOM) (<0.5 ppm of C) and from supply B with a high NOM concentration (7.9 ppm of C). Eukaryotic communities were studied using T-RFLP and clone-library analysis of partial 18S rRNA gene fragments and a Hartmannella vermiformis-specific q-PCR. In both supplies highly diverse eukaryotic communities were observed, including free-living protozoa, fungi, and metazoa. Sequences of protozoa clustered with Amoebozoa (10 OTUs), Cercozoa (39 OTUs), Choanozoa (26 OTUs), Ciliophora (29 OTUs), Euglenozoa (13 OTUs) Myzozoa (5 OTUs) and Stramenopiles (5 OTUs). A large variety of protozoa was present in both supplies, but the estimated values for protozoan richness did not differ significantly. H. vermiformis was observed in both supplies but was not a predominant protozoan. One OTU with the highest similarity to Acanthamoeba polyphaga, an opportunistic human pathogen and a host for undesired bacteria, was observed in supply A. The high level of NOM in supply B corresponded with an elevated level of active biomass and with elevated concentrations of H. vermiformis in distributed water. Hence, application of q-PCR may be promising in elucidating the relationship between drinking water quality and the presence of specific protozoa