Phylogenetic analysis of environmental Legionella pneumophila isolates from an endemic area (Alcoy, Spain).

Environmental surveillance of Legionella pneumophila is a key component of the control measures established in urban settlements to ensure water safety and quality, with the aim of minimizing and limiting opportunistic infections in humans. In this work, we present results on the detection and genetic characterization of these bacteria in the outbreak-recurrent region of Alcoy (Comunidad Valenciana, Spain) using water and biofilm samples. We were particularly interested in studying the presence and distribution of L. pneumophila in the absence of outbreak or sporadic cases of legionellosis and in comparing the efficacy of culturing from water samples with a biofilm-based detection procedure using molecular amplification. To this end, water samples were taken from 120 sites distributed all around the city and its surroundings, as well as 60 biofilm swabs from half of the sampling sites. L. pneumophila could be isolated from water in just 4 of the locations. Touchdown PCR was applied to DNA extracted from water and also biofilm swabs, as a rapid method for both routine and outbreak investigations. L. pneumophila was detected by this method in 14 of the sites in which both water and biofilms were taken, although 13 of them tested positive using only the biofilm samples. These results show a ten-fold increase in the success rate of Legionella detection over water samples. The application of this method to study the presence of L. pneumophila in the water-supply system and risk facilities of Alcoy revealed different strains distributed in different areas of the city. Sequence Type ST578, endemic in the area and responsible for most clinical cases, was detected in one of the sampling sites. The number of positive samples correlated with water temperature but not with chlorine levels. The direct analysis of biofilm swabs improves the detection rate and genetic characterization of L. pneumophila and can complement analyses based on bacterial culture.