Detection of Legionella spp. in natural and man-made water systems using standard guidelines

Abstract

Infections caused by Legionella spp. are considered at the present time, an emerging public health problem andare linked to high rates of mortality and morbidity, if not properly treated. In this study were analyzed 54 samples of waterfrom 8 counties at Northern Portugal, with the aim of obtaining a collection of strains of the genus Legionella and to characterizethem genetically and phenotypically. Another objective of this study was to evaluate the effectiveness of the techniqueof cultivation, a standard method according to International Organization for Standardization ISO 11731:1998, fordetection and enumeration of species of Legionella. For laboratory processing, after the filtration of samples (1 L), the filtratewas resuspended in sterile distilled water (5 ml). Heat treatment for selective inhibition of non-Legionella bacteria wasperformed. Subsequently, 100 l 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 agarfor verification. According to the procedure recommended by the standard method, only the colonies which grew in BCYEagar and not on blood agar were considered as suspected Legionella strains. The identification of these initially selectedcolonies was performed by sequencing the 16S rRNA gene, which revealed that none of the isolates were identified as belongingto the genus Legionella. However, through the ISO 11731:1998 they were interpreted as positive, correspondingtherefore to false-positive results. The methods used in this study allowed the isolation of a number of isolates (40), whichform an independent group of all genus of the family Chitinophagaceae outlined so far, and that by their phylogenetic distancemight be a genus not yet described and therefore a new species. The results obtained, highlighted the importance ofusing culture and genetic methods in parallel for the proper identification of microorganisms