Concentration and diversity of uncultured Legionella spp. in two unchlorinated drinking water supplies with different concentrations of natural organic matter

Abstract

Two unchlorinated drinking water supplies were investigated to assess the potential of water treatment and distribution systems to support the growth of Legionella spp. The treatment plant for supply A distributed treated groundwater with a low concentration (<0.5 ppm of C) of natural organic matter (NOM), and the treatment plant for supply B distributed treated groundwater with a high NOM concentration (8 ppm of C). In both supplies, the water temperature ranged from about 10°C after treatment to 18°C during distribution. The concentrations of Legionella spp. in distributed water, analyzed with quantitative PCR (Q-PCR), averaged 2.9 (± 1.9) × 10(2) cells liter(-1) in supply A and 2.5 (± 1.6) × 10(3) cells liter(-1) in supply B. No Legionella was observed with the culture method. A total of 346 clones (96 operational taxonomical units [OTUs] with ≥97% sequence similarity) were retrieved from water and biofilms of supply A and 251 (43 OTUs) from supply B. The estimation of the average value of total species richness (Chao1) in supply A (153) was clearly higher than that for supply B (58). In each supply, about 77% of the sequences showed <97% similarity to described species. Sequences related to L. pneumophila were only incidentally observed. The Legionella populations of the two supplies are divided into two distinct clusters based on distances in the phylogenetic tree as fractions of the branch length. Thus, a large variety of mostly yet-undescribed Legionella spp. proliferates in unchlorinated water supplies at temperatures below 18°C. The lowest concentration and greatest diversity were observed in the supply with the low NOM concentration.

FIG. 1.

Concentrations of Legionella spp. in water and in biofilms in two groundwater supplies. (A) Supply A (treated aerobic groundwater). (B) Supply B (treated anaerobic groundwater). Abbreviations: W1, W2, W3, and W4, raw-water wells; RW, raw water (combined); A, aeration; RSF, rapid sand filtration; PS, pellet softening; TW, treated water; D, water and biofilm sampled from distribution system. Bars show concentrations with standard deviations in duplicate samples, and for TW, with standard deviations in 8 (A) and 6 (B) samples collected over a period of 9 months. Open bars, water samples; solid bars, biofilm samples; stars, Legionella concentrations below the detection limit in water and in biofilm (D2).

FIG. 2.

Phylogenetic tree showing positions of the partial 16S rRNA gene sequences with ≥97% sequence similarities to described L. pneumophila sequences. A de novo parsimony tree was calculated in ARB with 82 16S rRNA gene sequences from described Legionella spp. (>1,400 bp) and three out-group sequences (Piscirickettsia salmonis, Wolbachia persica, and Coxiella burnetii). The clone sequences were added to the tree using the “Quick add” tool. A number of reference sequences were removed from the final tree to reduce its size.

FIG. 3.

UPGMA clusters of Legionella populations in aerobic groundwater, treated aerobic and anaerobic groundwater, and biofilm samples from pipe segments of the distribution network. The number of sequences that represents each environment is indicated next to the name. Calculated jackknife values (percentages) are indicated in each node. RW, raw water, TW, treated water; BF, biofilm from distribution system.