Presence and persistence of Legionella spp. in groundwater

Abstract

Groundwater samples (111) from six different boreholes located in two geographical areas were examined for the presence of legionellae over a 7-year period. The number of Legionella isolates detected was generally low. The colonization of the aquifers was not uniform, and the persistence of Legionella was independent of the hydraulic pumps and the plumbing system present in the borehole. A total of 374 isolates identified by fatty acid methyl ester analysis belonged to Legionella pneumophila, L. oakridgensis, L. sainthelensi, and L. londiniensis. In area 1, L. oakridgensis constituted the major population detected, exhibiting only one random amplified polymorphic DNA (RAPD)-PCR profile. L. sainthelensi strains were less frequently isolated and also displayed a single RAPD profile, while L. pneumophila was only sporadically detected. In contrast, L. pneumophila comprised the vast majority of the isolates in area 2 and exhibited six distinct RAPD patterns, indicating the presence of different genetic groups; three L. londiniensis RAPD types were also detected. Two of the L. pneumophila and one of the L. londiniensis RAPD types were persistent in this environment for at least 12 years. The genetic structure of L. pneumophila groundwater populations, inferred from rpoB and dotA gene sequences, was peculiar, since the majority of the isolates were allied in a discrete group different from the lineages containing most of the type and reference strains of the three subspecies of L. pneumophila. Furthermore, gene exchange events related to the dotA allele could be envisioned.

FIG. 1.

Geological formations in the vicinity of the sampling sites in central Portugal. (A) Sampling sites in area 1. (B) Sampling sites in area 2.

FIG. 2.

Agarose gel electrophoresis of RAPD types obtained from representative isolates of Legionella spp. detected in groundwater and biofilm samples from areas 1 and 2 and respective type strains. M, Marker XIV (Roche Molecular Biochemical). Values to the left are in base pairs.

FIG. 3.

Comparison of RAPD types obtained from strains isolated in 1991 and the more persistent RAPD types from 2003 in area 2. M, Marker XIV (Roche Molecular Biochemical). Values to the left are in base pairs.

FIG. 4.

Phylogenetic dendrograms based on analyses of partial sequences of dotA (A) and rpoB (B) of the groundwater isolates and of type and references strains of L. pneumophila. The trees were created using the neighbor-joining method. Isolates and the respective RAPD types characterized in this study are indicated in boldface. Branches supported by values of >50% in the bootstrap analysis (1,000 replications) are indicated. Scale bar, 5 inferred nucleotide substitutions per 100 nucleotides.