INVESTIGATION OF LEGIONELLA SPECIES IN TEHRANíS HOSPITAL WATER SUPPLIES
MOHAMMAD RAFIEE, JAHANGIRI-RAD MAHSA
ABSTRACT. Ė Investigation of Legionella species in Tehranís 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 hospitalsí 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