Detection of protozoan hosts for Legionella pneumophila in engineered water systems by using a biofilm batch test

Abstract

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.

FIG. 1.

Maximum concentrations of L. pneumophila (mip gene copies cm⁻²), Legionella spp. (GU cm⁻²), and H. vermiformis (cells cm⁻²) in the biofilm on PE-Xa in the BBT flasks with biomass from a limestone filter bed in treated water of supply A incubated at 37°C for 20 days. Initial concentrations of L. pneumophila (about 2 log units of mip gene copies cm⁻²) and H. vermiformis (about 3 log units of cells cm⁻²) are converted from units liter⁻¹ to units cm⁻². Abbreviations: T-I and T-II, blank test flasks (no inoculation); T+Hv-I and T+Hv-II, duplicate test flasks inoculated with H. vermiformis; T+Lp-I and T+Lp-II, duplicate test flasks inoculated with L. pneumophila; T+Hv+Lp-I and T+Hv+Lp-II, duplicate test flasks inoculated with L. pneumophila and H. vermiformis (controls). Error bars indicate standard deviations of the analysis.

FIG. 2.

Growth of inoculated L. pneumophila in two biofilm batch test flasks with water from cooling tower 1 during incubation at 37°C for 23 days. Symbols: ○, L. pneumophila (mip gene copies liter⁻¹); ▴, Legionella spp. (genome units [GU] liter⁻¹); ▪, H. vermiformis (cells liter⁻¹). The detection limit for H. vermiformis is 20 cells liter⁻¹. Error bars indicate standard deviations of the analysis.

FIG. 3.

UPGMA (unweighted-pair group method using average linkages) dendrogram of T-RFLP fingerprints of the BBT flasks with (i) biomass from a limestone filter bed of supply A (biomass filter A), (ii) treated sewage, and (iii) water from warm tap water installation B1 in distribution area of supply B (warm tap water B) at day 0 and after 20 days of incubation at 37°C with L. pneumophila (+ Lp). Sample volumes, 50 to 200 ml of the planktonic sample and 50 to 100 ml of the biofilm suspensions. The numbers above the dendrogram represent percent similarity, and those above the fingerprints represent fragment length (number of nucleotides).