Multiplex polymerase chain reaction of genetic markers for detection of potentially pathogenic environmental isolates.

Background & Objectives: Genomic constitution of the bacterium Legionella pneumophila plays an important role in providing them a pathogenic potential. Here, we report the standardization and application of multiplex polymerase chain reaction (PCR) for the detection of molecular markers of pathogenic potential in L. pneumophila in hospital environment.

Methods: Culture of the standard strains of L. pneumophila was performed in buffered charcoal-yeast extract agar with L-cysteine at p H 6.9. Primers were designed for multiplex PCR, and standardization for the detection of five markers annotated to L. pneumophila plasmid pLPP (11A2), lipopolysaccharide synthesis (19H4), CMP-N-acetylneuraminic acid synthetase (10B12), conjugative coupling factor (24B1) and hypothetical protein (8D6) was done. A total of 195 water samples and 200 swabs were collected from the hospital environment. The bacterium was isolated from the hospital environment by culture and confirmed by 16S rRNA gene PCR and restriction enzyme analysis. A total of 45 L. pneumophila isolates were studied using the standardized multiplex PCR.

Results: The PCR was sensitive to detect 0.1 ng/μl DNA and specific for the two standard strains used in the study. Of the 45 hospital isolates tested, 11 isolates had four markers, 12 isolates had three markers, 10 isolates had two markers, nine isolates had one marker and three isolates had none of the markers. None of the isolates had all the five markers.

Interpretation & Conclusions: The findings of this study showed the presence of gene markers of pathogenic potential of the bacterium L. pneumophila. However, the genomic constitution of the environmental isolates should be correlated with clinical isolates to prove their pathogenic potential. Rapid diagnostic methods such as multiplex PCR reported here, for elucidating gene markers, could help in future epidemiological studies of bacterium L. pneumophila.