Detection of Legionella spp. in Natural and Man-made Water Systems Using Standard Guidelines

Infections caused by Legionella spp. are considered at the present time, an emerging public health problem and are linked to high rates of mortality and morbidity, if not properly treated. In this study were analyzed 54 samples of water from 8 counties at Northern Portugal, with the aim of obtaining a collection of strains of the genus Legionella and to characterize them genetically and phenotypically. Another objective of this study was to evaluate the effectiveness of the technique of cultivation, a standard method according to International Organization for Standardization ISO 11731:1998, for detection and enumeration of species of Legionella. For laboratory processing, after the filtration of samples (1 L), the filtrate was resuspended in sterile distilled water (5 ml). Heat treatment for selective inhibition of non-Legionella bacteria was performed. Subsequently, 100 μl of the suspension was spread in GVPC selective agar medium, and incubated (7 to 10 days) at 37 ℃. Colonies that were morphologically characteristic of the genus were sub-cultured onto BCYE agar and blood agar for verification. According to the procedure recommended by the standard method, only the colonies which grew in BCYE agar and not on blood agar were considered as suspected Legionella strains. The identification of these initially selected colonies was performed by sequencing the 16S rRNA gene, which revealed that none of the isolates were identified as belonging to the genus Legionella. However, through the ISO 11731:1998 they were interpreted as positive, corresponding therefore to “false-positive” results. The methods used in this study allowed the isolation of a number of isolates (40), which form an independent group of all genus of the family Chitinophagaceae outlined so far, and that by their phylogenetic distance might be a genus not yet described and therefore a new species. The results obtained, highlighted the importance of using culture and genetic methods in parallel for the proper identification of microorganisms.