Efficacy of PCR Analysis of Mip, Doth and Gspd Genes with Culture in Detection of Legionella pneumophila.

Dear Editor-in-Chief

Legionella microorganism is ubiquitous and found worldwide naturally in rivers, streams, springs of hot water, swimming pools, tanks, water piping networks, cooling tower and conditioning systems (1). This bacterium causes sporadic and epidemic cases of community-acquired pneumonia (CAP) in healthy and immunocompromised from hospital or community settings (2). Studies showed that 3% to 8% of all CAP are possibly caused by Legionella spp. where 85% of those caused by L. pneumophila (3). Two independent clinical diseases caused by Legionella species include; legionellosis that is a severe form of pneumonia and another one is Pontiac fever; a self-limiting flu-like disease (4). We aimed to investigate the efficacy of PCR analysis of mip, dotH and gspD genes with culture in the detection of L. pneumophila.

In this cross-sectional study during 2016, 100 samples (50 of clinical samples and 50 samples from hospital water) were collected. Detection and identification of Legionella isolates was performed using microbiological methods and biochemical tests. Samples treated with a solution of N HCL-KCL2 and then incubated in 56 °C for 12 min. Then, DNA of them was extracted. And PCR technique was performed for the detection of genes. To design primers of selected genes, all genome sequences were identified in the genome databases, then assembled and analyzed, and primers designed with Gene Runner software after design, selected primers were blasted by BLast N to compare the sequence of primers with existing GenBank records. The primers sequences were as follows; F-t4ss:5′-GTGTGGTGTAGGCTGGTTTG-3′, R-t4ss: 5′-CTAACCCAGAAGTGCCGATT-3′, F-mip: 5′-AAAGGCATGCAAGACGCTAT-3′; R-mip: 5′-GTATCCGATTTTCCGGGTTT-3, F-16srRNA: 5′-AGGGTTGATAGGTTAAGAGC-3′, R-16srRNA: 5′-CCAACAGCTAGTTGACATCG-3′; F-t2ss: 5′- GGGCATTAGTGGCCTTAGAA-3′, R-t2ss: 5′CTCCACGAGGTGACGATATG-3′. Then data statistically analyzed using SPSS (Chicago, IL, USA) software trough Chi-square test.

Based on the results of culture, 14(14%) isolates of Legionella were recovered from clinical and water samples. PCR results showed 64(64%) out of 100 samples were positive for each of mip and dotH genes. Of these 64 positive samples for dotH genes (24 and 40 cases belonged to the clinical and water samples, respectively). Among the 64 samples were positive for mip gene, 42 and 22 cases belonged to the water and clinical samples, respectively (Fig. 1). Furthermore, 53(53%) of samples were positive for the gspD gene, of which 23(43.4%) of samples were from clinical and remaining from water samples.

Fig. 1:

A)PCR image of mip gene of Legionella spp., on the 1% gel electrophoresis. Line M: Marker 50 bp, line 1: It corresponds to positive control, line 2: It is related with negative control and line 3, 4 and 5: Product size 242 bp of mip gene in tested samples. B)PCR image of dotH and gspD genes of Legionella spp., on the 1% gel electrophoresis. Line1: Marker 50 bp, well 2: Positive control for gspD gene, well 3: environmental sample for gspD gene, well 4: negative control, well 5: Clinical specimen for gspD gene, well 6: positive control for dotH gene and wells 7 and 8: Environmental samples for the dotH gene

There were some limitations to separate Legionella from samples by culture include; a long incubation period and Legionella growth is overshadowed by fast-growing organisms (5), as well as presence of living Legionella that doesn’t have the power to grow on the media, so, all species of Legionella are not detectable by culture (6). Therefore, it is imperative that despite the importance and high sensitivity of culture in isolation of this organism, in addition to the culture, PCR technique can be also used to detecting this bacterium. For the first time in this study dotH and gspD genes was used alongside with mip gene by PCR technique for diagnosis of Legionella, and according to the obtained findings, sensitivity rate of two genes was comparable, so can use of them as promising genes in rapid detection of Legionella from different samples.

Results showed the prevalence of three genes; mip, dotH and gspD is high using PCR, so can use of these genes in PCR as a rapid detection method accompanying with culture for diagnosis of Legionella.