Investigation Of Legionella Species In Tehran’s Hospital Water Supplies
The present work was performed to investigate the presence of Legionella spp. and its common species in hospital
water supplies. Considering the drawback of culture method, polymerase chain reaction (PCR) assays were developed to
detect the gene 16S rRNA regardless of the bacterial serotype. Four well-established DNA extraction protocols (freeze &
thaw and phenol-chloroform as two manual protocols and two commercial kits) were examined and critiqued to release
DNA from bacterial cells. A total of 45 samples were collected from seven distinct hospitals’ sites during a period of 10
months. The PCR assay was exploited to amplify a 654-bp fragment of the 16S rRNA gene. Legionella were detected in 13
samples (28.9%) by all of the methods applied for DNA extraction. Considerable differences were noted in the yield of
extracted nucleic acids. Legionella were not detected in any of the samples when DNA extraction by freeze & thaw was
used. Omitting this method and comparing manual protocol with commercial kits, Kappa coefficient was calculated as 0.619
with p < 0.05. Although no meaningful differences were found between the kits, DNA extraction with Bioneer kit displayed
a higher sensitivity than classical Qiagen. Showerheads and cold-water taps were the most and least contaminated sources
with 55.5 and 9 percent positive samples, respectively. Moreover two positive samples were identified for species by DNA
sequencing and submitted to the Gene Bank database with accession Nos. FJ480932 and FJ480933.
Keywords- Legionella, Hospital Water Supplies, DNA Extraction, PCR, 16S RRNA.