Free-living protozoan communities in water supplies may include hosts for Legionella pneumophila and other undesired bacteria, as well as pathogens. This study aimed at identifying free-living protozoa in two unchlorinated groundwater supplies, using cultivation-independent molecular approaches. For this purpose, samples (<20°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 terminal restriction fragment length polymorphism and clone library analyses of partial 18S rRNA gene fragments and a Hartmannella vermiformis-specific quantitative PCR (qPCR). In both supplies, highly diverse eukaryotic communities were observed, including free-living protozoa, fungi, and metazoa. Sequences of protozoa clustered with Amoebozoa (10 operational taxonomic units [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 were 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, the application of qPCR may be promising in elucidating the relationship between drinking water quality and the presence of specific protozoa.Free-living protozoa are ubiquitous in natural freshwater environments (7,38,51,71) but also proliferate in engineered water systems, including water treatment systems (3,47,70), distribution systems (6, 75), and tap water installations inside buildings (54, 69). Concentrations of protozoa, determined using cultivation methods and microscopy, range from Ͻ1 to 10 4 cells liter Ϫ1 in treated water (3,47,70,75) and from Ͻ1 to 7 ϫ 10 5 cells liter Ϫ1 in distribution systems (6,61,64,75). Genera of free-living protozoa commonly observed in these systems and in tap water installations include Acanthamoeba,

A real-time PCR-based method targeting the 18S rRNA gene was developed for the quantitative detection of Hartmannella vermiformis, a free-living amoeba which is a potential host for Legionella pneumophila in warm water systems and cooling towers. The detection specificity was validated using genomic DNA of the closely related amoeba Hartmannella abertawensis as a negative control and sequence analysis of amplified products from environmental samples. Real-time PCR detection of serially diluted DNA extracted from H. vermiformis was linear for microscopic cell counts between 1.14 ؋ 10 ؊1 and 1.14 ؋ 10 4 cells per PCR. The genome of H. vermiformis harbors multiple copies of the 18S rRNA gene, and an average number (with standard error) of 1,330 ؎ 127 copies per cell was derived from real-time PCR calibration curves for cell suspensions and plasmid DNA. No significant differences were observed between the 18S rRNA gene copy numbers for trophozoites and cysts of strain ATCC 50237 or between the copy numbers for this strain and strain KWR-1. The developed method was applied to water samples (200 ml) collected from a variety of lakes and rivers serving as sources for drinking water production in The Netherlands. Detectable populations were found in 21 of the 28 samples, with concentrations ranging from 5 to 75 cells/liter. A high degree of similarity (>98%) was observed between sequences of clones originating from the different surface waters and between these clones and the reference strains. Hence, H. vermiformis, which is highly similar to strains serving as hosts for L. pneumophila, is a common component of the microbial community in fresh surface water.

The study whose results are presented here aimed at identifying free-living protozoa (FLP) and conditions favoring the growth of these organisms and cultivable Legionella spp. in drinking water supplies in a tropical region. Treated and distributed water (؎30°C) of the water supplies of three Caribbean islands were sampled and investigated with molecular techniques, based on the 18S rRNA gene. The protozoan host Hartmannella vermiformis and cultivable Legionella pneumophila were observed in all three supplies. Operational taxonomic units (OTUs) with the highest similarity to the potential or candidate hosts Acanthamoeba spp., Echinamoeba exundans, E. thermarum, and an Neoparamoeba sp. were detected as well. In total, 59 OTUs of FLP were identified. The estimated protozoan richness did not differ significantly between the three supplies. In supply CA-1, the concentration of H. vermiformis correlated with the concentration of Legionella spp. and clones related to Amoebozoa predominated (82%) in the protozoan community. These observations, the low turbidity In tropical regions, the water temperature in drinking water distribution system is permanently about 30°C (4). In these regions, free-living protozoa (FLP), serving as hosts for pathogenic bacteria, including Acanthamoeba spp. (1, 38), Hartmannella spp. (37), and Naegleria spp. (38, 49), have been observed in surface water, wastewater, cooling towers, and drinking water (3,5,19,45). Certain FLP with pathogenic properties, viz. Acanthamoeba spp. (9, 21), Balamuthia mandrillaris (54), and Naegleria fowleri (59), can proliferate in drinking water-related biofilms at elevated temperatures (36). In addition, Legionella pneumophila, the main etiologic agent of Legionnaires' disease (12), which proliferates in freshwater at temperatures above 25°C (57), is frequently observed in these environments (14,31,39).FLP in aquatic environments feed on bacteria, fungi, other protozoa, and organic detritus in biofilms and sediments or in the planktonic phase (32). The abundance of prey organisms and detritus depends on the water composition and the hydraulic conditions in distribution systems, which therefore also affect both the FLP abundance and community composition (52, 55). Most information on community composition and abundance of FLP in freshwater environments has been obtained by using cultivation methods and microscopy. Recently, however, the presence and identities of such organisms in drinking water supplies in temperate regions have been studied by using molecular methods for detection and identification (33, 51). In two groundwater supplies in the Netherlands a total of 127 operational taxonomic units (OTUs) of FLP were identified based on their 18S rRNA gene sequences. FLP, mostly pathogens, have been characterized in only a few studies in tropical regions (3,5,19). In recent reviews it was concluded that more research is needed to determine which factors favor the growth of these organisms in water supplies (47,48).Cases of Legionnaires' disease have been reporte...

Legionella pneumophila proliferates in aquatic habitats within free-living protozoa, 17 species of which have been identified as hosts by using in vitro experiments. The present study aimed at identifying protozoan hosts for L. pneumophila by using a biofilm batch test (BBT). Samples (600 ml) collected from 21 engineered freshwater systems, with added polyethylene cylinders to promote biofilm formation, were inoculated with L. pneumophila and subsequently incubated at 37°C for 20 days. Growth of L. pneumophila was observed in 16 of 18 water types when the host protozoan Hartmannella vermiformis was added. Twelve of the tested water types supported growth of L. pneumophila or indigenous Legionella anisa without added H. vermiformis. In 12 of 19 BBT flasks H. vermiformis was indicated as a host, based on the ratio between maximum concentrations of L. pneumophila and H. vermiformis, determined with quantitative PCR (Q-PCR), and the composition of clone libraries of partial 18S rRNA gene fragments. Analyses of 609 eukaryotic clones from the BBTs revealed that 68 operational taxonomic units (OTUs) showed the highest similarity to free-living protozoa. Forty percent of the sequences clustering with protozoa showed >99.5% similarity to H. vermiformis. None of the other protozoa serving as hosts in in vitro studies were detected in the BBTs. In several tests with growth of L. pneumophila, the protozoa Diphylleia rotans, Echinamoeba thermarum, and Neoparamoeba sp. were identified as candidate hosts. In vitro studies are needed to confirm their role as hosts for L. pneumophila. Unidentified protozoa were implicated as hosts for uncultured Legionella spp. grown in BBT flasks at 15°C.Legionella pneumophila, the causative agent of Legionnaires' disease, is a common inhabitant of natural freshwater environments and human-made water systems, including cooling towers, whirlpools, air-conditioning systems, and installations for warm tap water (14). In the aquatic environment L. pneumophila proliferates within certain free-living protozoa, which serve as its hosts (15,30,59). Environmental factors favoring the growth and survival of L. pneumophila in freshwater systems include a water temperature between 20°C and 45°C (41, 60) and the presence of biofilms and sediments on which the protozoan hosts can graze (30,41,56).Rowbotham (44) was the first to report the growth of L. pneumophila within free-living amoebae, which belonged to the genera Acanthamoeba and Naegleria. In vitro studies with cocultures have revealed that 14 species of amoebae, viz., Acanthamoeba spp. (1, 35, A number of the free-living protozoa mentioned above and others, e.g., Vannella spp. and Saccamoeba spp., have been observed in aquatic environments from which L. pneumophila was cultivated or in which it was detected with PCR (4, 42, 51, 52). However, it remains unknown which of these protozoa actually serve as hosts for L. pneumophila in the aquatic environment, including human-made water systems. Moreover, it cannot be excluded that free-living protozoa oth...

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