Occurrence and genetic diversity of uncultured Legionella spp. in drinking water treated at temperatures below 15 degrees C

Abstract

Representatives of the genus Legionella were detected by use of a real-time PCR method in all water samples collected directly after treatment from 16 surface water (SW) supplies prior to postdisinfection and from 81 groundwater (GW) supplies. Legionella concentrations ranged from 1.1 x 10(3) to 7.8 x 10(5) cells liter(-1) and were significantly higher in SW treated with multiple barriers at 4 degrees C than in GW treated at 9 to 12 degrees C with aeration and filtration but without chemical disinfection. No Legionellae (<50 CFU liter(-1)) were detected in treated water by the culture method. Legionella was also observed in untreated SW and in untreated aerobic and anaerobic GW. Filtration processes in SW and GW treatment had little effect or increased the Legionella concentration, but ozonation in SW treatment caused about 1-log-unit reduction. A phylogenetic analysis of 16S rRNA gene sequences of 202 clones, obtained from a selection of samples, showed a high similarity (>91%) with Legionella sequences in the GenBank database. A total of 40 (33%) of the 16S rRNA gene sequences obtained from treated water were identified as described Legionella species and types, including L. bozemanii, L. worsleiensis, Legionella-like amoebal pathogen types, L. quateirensis, L. waltersii, and L. pneumophila. 16S rRNA gene sequences with a similarity of below 97% from described species were positioned all over the phylogenetic tree of Legionella. Hence, a large diversity of yet-uncultured Legionellae are common members of the microbial communities in SW and GW treated at water temperatures of below 15 degrees C.

FIG. 1.

Frequency distributions of Legionella concentrations (N, cells/16S rRNA gene copies per liter) in treated water from 97 treatment plants analyzed by a real-time PCR method using the 16S rRNA gene-targeting primers LEG-225 and LEG-858. GW-an, groundwater supply with anoxic source water; GW-aer, groundwater supply with aerobic source water; SW, surface water supply.

FIG. 2.

Phylogenetic trees showing the positions of the 202 16S rRNA gene sequences of the partial 16S RNA gene amplified using Legionella-specific primers LEG-225 and LEG-858. The subtree in panel B includes mostly LLAP-related 16S rRNA gene sequences, and that in panel C contains sequences mostly originating from SW and samples from treated SW. The 16S rRNA gene sequences shown in the trees are derived from seven SW samples (S1 to S7), three storage basins (Sb1 to Sb3), eight SW treatment plants (Tsw1 to Tsw8), ten anaerobic GW treatment plants (Tang1 to Tang10), and eight aerobic GW treatment plants (Tag1 to Tag8). The trees were constructed by using the parsimony tool with local optimization. The bar represents percentage sequence difference. Asterisks indicate 16S rRNA gene sequences from slow sand filters (13). Origin of 16S rRNA gene sequences included in groups (≥99% similarity): 1, S2- and S7-1; 2, Tang10-3, Tag3-3, and Sb3-10; 3, Tang8-5, Tag4-1, Tag4-2, and Tag4-5; 4, Tag3-1, Tag3-2, and Tag3-5; 5, Tsw5-3 and Tang6-2; 6, Tang10-4 and Tang7-3; 7, Tsw8-1 and Tsw8-2; 8, Tang8-1, Tang8-3, and Tang8-4; 9, Tang8-2, Tsw1-8, and Tang4-5; 10, Tsw4-2, Tsw4-4, and Tsw4-5; 11, Tag2-4, Tag2-5, Tag6-1, and Tang6-3; 12, Tsw7-4, Tag3-4, and Sb1-2; 13, Tsw5-2, Tsw5-4, Tsw5-5, and Tsw4-3; 14, Sb3-3 and Sb3-6; 15, Tang9-3, Tag5-3, and Tang2-3; 16, Tang3-1, Tang5-1, Tang5-3, and Tang5-5; 17, Tang10-5 and Sb3-1; 18, S2-1 and S2-6; 19, Tang9-2, Tang3-2, Tang3-3, and Tang3-5; 20, Sb3-2 and Sb3-9; 21, Tang5-4, Tang1-2, and Tang1-5; 22, S4-1, Tang4-3, and S4-8; 23, S7-3 and Tsw1-5; 24, Tsw3-4 and Tag1-4; 25, Sb2-2 and Sb2-6; 26, S5-5, S6-2, S4-9, and S2-9; 27, Tsw6-1, Tsw6-6, and Tsw6-9; 28, Tsw6-7, S1-2, S1-3, and Tsw6-8; 29, Tsw6-3 and Tsw6-5; 30, S5-6 and S5-8; 31, Tsw1-1, Tsw1-4, and Tsw1-7; 32, S2-4 and S2-5; 33, Tsw8-3 and Tsw8-4; 34, S2-3 and S7-9; 35, S4-5, S4-3, and S4-7; 36, Tsw5-1, Tang9-1, and Tsw3-3; 37, Sb1-8 and Sb1-10; 38, Tag5-1 and Tag5-2; 39, S6-4 and S6-5; 40, Tsw7-1, Tsw7-2, and Tsw7-3; 41, Tsw7-5, Tsw7-7, and Tsw7-9; 42, Tsw2-1 and Tsw2-2; 43, Tsw2-3 and Tag1-1; and 44, Sb2-1, Sb2-4, and Sb2-7.

FIG. 2.

Phylogenetic trees showing the positions of the 202 16S rRNA gene sequences of the partial 16S RNA gene amplified using Legionella-specific primers LEG-225 and LEG-858. The subtree in panel B includes mostly LLAP-related 16S rRNA gene sequences, and that in panel C contains sequences mostly originating from SW and samples from treated SW. The 16S rRNA gene sequences shown in the trees are derived from seven SW samples (S1 to S7), three storage basins (Sb1 to Sb3), eight SW treatment plants (Tsw1 to Tsw8), ten anaerobic GW treatment plants (Tang1 to Tang10), and eight aerobic GW treatment plants (Tag1 to Tag8). The trees were constructed by using the parsimony tool with local optimization. The bar represents percentage sequence difference. Asterisks indicate 16S rRNA gene sequences from slow sand filters (13). Origin of 16S rRNA gene sequences included in groups (≥99% similarity): 1, S2- and S7-1; 2, Tang10-3, Tag3-3, and Sb3-10; 3, Tang8-5, Tag4-1, Tag4-2, and Tag4-5; 4, Tag3-1, Tag3-2, and Tag3-5; 5, Tsw5-3 and Tang6-2; 6, Tang10-4 and Tang7-3; 7, Tsw8-1 and Tsw8-2; 8, Tang8-1, Tang8-3, and Tang8-4; 9, Tang8-2, Tsw1-8, and Tang4-5; 10, Tsw4-2, Tsw4-4, and Tsw4-5; 11, Tag2-4, Tag2-5, Tag6-1, and Tang6-3; 12, Tsw7-4, Tag3-4, and Sb1-2; 13, Tsw5-2, Tsw5-4, Tsw5-5, and Tsw4-3; 14, Sb3-3 and Sb3-6; 15, Tang9-3, Tag5-3, and Tang2-3; 16, Tang3-1, Tang5-1, Tang5-3, and Tang5-5; 17, Tang10-5 and Sb3-1; 18, S2-1 and S2-6; 19, Tang9-2, Tang3-2, Tang3-3, and Tang3-5; 20, Sb3-2 and Sb3-9; 21, Tang5-4, Tang1-2, and Tang1-5; 22, S4-1, Tang4-3, and S4-8; 23, S7-3 and Tsw1-5; 24, Tsw3-4 and Tag1-4; 25, Sb2-2 and Sb2-6; 26, S5-5, S6-2, S4-9, and S2-9; 27, Tsw6-1, Tsw6-6, and Tsw6-9; 28, Tsw6-7, S1-2, S1-3, and Tsw6-8; 29, Tsw6-3 and Tsw6-5; 30, S5-6 and S5-8; 31, Tsw1-1, Tsw1-4, and Tsw1-7; 32, S2-4 and S2-5; 33, Tsw8-3 and Tsw8-4; 34, S2-3 and S7-9; 35, S4-5, S4-3, and S4-7; 36, Tsw5-1, Tang9-1, and Tsw3-3; 37, Sb1-8 and Sb1-10; 38, Tag5-1 and Tag5-2; 39, S6-4 and S6-5; 40, Tsw7-1, Tsw7-2, and Tsw7-3; 41, Tsw7-5, Tsw7-7, and Tsw7-9; 42, Tsw2-1 and Tsw2-2; 43, Tsw2-3 and Tag1-1; and 44, Sb2-1, Sb2-4, and Sb2-7.