In this study, we investigated the distribution of Legionella species in water cooling towers located in different parts of Malaysia to obtain information that may inform public health policies for the prevention of legionellosis. A total of 20 water samples were collected from 11 cooling towers located in three different states in east, west and south Malaysia. The samples were concentrated by filtration and treated with an acid buffer before plating on to BCYE agar. Legionella viable counts in these samples ranged from 100 to 2,000 CFU ml(-1); 28 isolates from the 24 samples were examined by latex agglutination as well as 16S rRNA and rpoB PCR-DNA sequencing. These isolates were identified as Legionella pneumophila serogroup 1 (35.7%), L. pneumophila serogroup 2-14 (39%), L. pneumophila non-groupable (10.7%), L. busanensis, L. gormanii, L. anisa and L. gresilensis. L. pneumophila was clearly the predominant species at all sampling sites. Repeat sampling from the same cooling tower and testing different colonies from the same water sample showed concurrent colonization by different serogroups and different species of Legionella in some of the cooling towers.
Male-specific coliphages are often used as indicators of contamination by enteric viruses. These phages can be detected in water samples by plaque assays and by polymerase chain reaction. In this study, the M13 coliphage was used to develop a real-time PCR assay for the detection of male-specific DNA coliphages. The real-time PCR was found to have a reaction efficiency of 1.45 and detection limit of 10(-3) plaque forming units per reaction mix. Repeated amplification and melting curve analyses demonstrated high specificity and reproducibility of the real-time assay. Quantitative detection with the real-time PCR should allow rapid assessment of the level of viral contamination in water.
The detection of Legionella pneumophila in environmental and clinical samples is frequently performed by PCR amplification of the mip and/or 16S rRNA genes. Combined with DNA sequencing, these two genetic loci can be used to distinguish different species of Legionella and identify L. pneumophila. However, the recent Legionella genome sequences have opened up hundreds of possibilities for the development of new molecular targets for detection and diagnosis. Ongoing comparative genomics has the potential to fine tune the identification of Legionella species and serogroups by combining specific and general genetic targets. For example, the coincident detection of LPS biosynthesis genes and virulence genes may allow the differentiation of both pathogen and serogroup without the need for nucleotide sequencing. We tested this idea using data derived from a previous genomic subtractive hybridization we performed between L. pneumophila serogroup 1 and L. micdadei. Although not yet formally tested, these targets serve as an example of how comparative genomics has the potential to improve the scope and accuracy of Legionella molecular detection if embraced by laboratories undertaking Legionella surveillance.