Detection and identification of Legionella species in hospital water supplies through polymerase chain reaction (16S rRNA)

Legionella spp. are important waterborne pathogens that are normally transmitted through aerosols. The present
work was conducted to investigate the presence of Legionella spp. and its common species in hospital water
supplies. Considering the limitations of culture method, polymerase chain reaction (PCR) assays were developed to
detect the gene 16S rRNA irrespective of the bacterial serotype. Four well-established DNA extraction protocols
(freeze & thaw and phenol-chloroform as two manual protocols and two commercial kits) were tested and
evaluated 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 used 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. Significant
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. Excluding 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 exhibited 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. The results obtained showed that despite
the advantages of molecular assays in Legionella tracing in environmental sources, the use of optimised DNA
extraction methods is critical.