Population structure and recombination in environmental isolates of Legionella pneumophila

Legionella pneumophila is a water-borne bacteria responsible for most cases of legionellosis, an emerging disease with an increasing incidence in industrialized countries. Although early analysis based on multilocus enzyme electrophoresis (MLEE) described the population structure of this species as clonal, more recent reports have suggested that recombination also contributes to shaping variation across its genome. We report here the results of analysing the nucleotide sequences of 19 loci in 31 environmental samples of L. pneumophila from a small Spanish region (near Alcoi, province of Alicante) where legionellosis has become almost endemic. We analysed the six loci currently incorporated to the sequence-based typing scheme developed by European Working Group for Legionella Infections (EWGLI) for L. pneumophila and 13 intergenic regions, for which we developed primers anchored in flanking, conserved genes. Our results show that recombination among natural isolates of this species is a common phenomenon, as 20 of the 31 isolates contained at least one locus in which recombination was revealed by at least three different methods. The mapping of the recombination events on the maximum likelihood tree of the concatenate sequence of the 19 loci indicated that at least nine independent recombination events might explain the observed distribution of recombinant loci among isolates. In consequence, we have shown that recombination in L. pneumophila is much more frequent than previously considered and that it does not seem to be restricted to already described pathogenicity islands or other genome constituents which provide it with a high plasticity.