Isolation and Identification of Legionella spp. in environmental water sources based on macrophage infectivity potentiator (mip) gene sequencing in southwest Iran

Legionella species are widespread in natural water sources and man-made aqueous environments, as well as fresh-water. The present study was conducted owing to the lack of research regarding the prevalence of Legionella spp in the water sources of Ahvaz city in southwest Iran. In this study the macrophage infectivity potentiator (mip) gene sequencing was used for identification of various Legionella species isolated from different water sources. In this study, 144 water samples were collected and inoculated on the buffered charcoal-yeast extract (BCYE) agar and modified Wadowsky-Yee (MWY) medium. The DNA was extracted from positive cultures. The Legionella species were confirmed by amplifying a 654 bp fragment of the 16S rRNA gene. The mip gene of all isolates were amplified by PCR and purified for sequencing. The mip gene sequences were analyzed by jPHYDIT software version 1. The results showed a 13.9% (20/144) prevalence of Legionella spp. in water sources of Ahvaz city, southwest Iran. Analyzing of the mip gene sequences showed, out of 20 Legionella isolates, 13 isolates (54.1%) were positive for L. pneumophila, 5 isolates (20.8%) were positive for L. worsleinsis, one isolates for each one of L. dumoffi and L. fairfieldensis, (4.1%). According to our research, the occurrence of Legionella spp in water sources could be a hazard for the health systems especially in the hospitals. The regular monitoring of these water sources by health planners may therefore be useful for decreasing the risk for Legionella spp. infections.


Introduction
Legionella species are widespread in natural water sources and man-made aqueous environments, as well as in fresh-water and wet soils [1]. These bacteria spread by Legionellacontaining water aerosols and cause infection in exposed people who have risk factors such as smoking, age more than 50 years, weakened immune system, diabetes, cancer, or chronic lung disease [2,3]. In most cases, Legionnaires' disease (LD), the sever pneumonic form of disease, is caused by Legionella pneumophila serogroup 1 (L. pneumophila SG-1), while the Pontiac fever, milder form of the disease, occurs by other non-pneumophila Legionella species [3]. The presence of Legionella spp. is favored by the attendance of algae and protozoa (amoebas and ciliates), in which they multiply intracellularly [4].
Legionella spp. have increased tolerance to chlorination, so they could enter potable hot water systems and multiply in different water sources, including cooling towers, whirlpool spas, hot tubes ,fishponds, shower heads, , holding tanks and respiratory ventilators [1,5]. Although some of studies have indicated that remaining more than 2 mg/l of chlorine in water, could eradicate freeliving Legionella [6], however, L. pneumophila living within biofilms is more resistant to annihilation through chlorine [7].
Although the culture method remains among the detection tools of Legionella in water sources, the excessive growth of accompanying living organisms or the conversion of Legionella cells to a viable but nonculturable form (VBNC) may affect the culture's results [8,9]. Thus, an effective assay for detection of Legionella species appears to be essential. It should be emphasized that detection of Legionella on the genus level as well as differentiation between L. pneumophila and non-L. pneumophila species is highly important for hazard prediction and the elimination of Legionella [8,10].
So far, several molecular methods that target Legionella spp. in water sources, have been reported, including L. pneumophila PAL antigen, and the polymerase chain reaction (PCR) assays targeting the 5S rRNA gene, the 23S-5S spacer region, the 16S rRNA gene, and the macrophage infectivity potentiator (mip) gene [8,[11][12][13]. Among the aforementioned methods, PCR amplification and sequencing of the mip gene seems to be a reliable method, which has known as a standard method [14]. In this field, the database, established by the members of the European Working Group for Legionella Infections (EWGLI), is freely accessible, which contains the data from all valid described species [15].
The present study aimed to evaluate the prevalence of Legionella spp. in water samples and to determine the species by mip gene sequencing in Ahvaz, southwest Iran.

Ethical Consideration
Not applicable.

Collection, preparation, and culture of water samples
In this study, 144 water samples were collected from different water supplies in 500 mL sterile plastic containers without neutralizing agent, from May to July 2014. Before sampling, the water sample was mixed with the sediment. The water samples were taken from the 5 teaching hospitals of Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran. However, some of the water sources were located out of hospital environments.
In the laboratory, each water sample was centrifuged at 3000 g for 30 min at 25 ℃. The 0.5 mL of sediment was treated by 4.5 mL washing buffer acid (HCL/KCL, pH 2.2) for 4 min. After mixing by wortex, 200 µL of the suspension was inoculated on two non-selective and selective media, buffered charcoal-yeast extract (BCYE) agar, and modified Wadowsky-Yee (MWY) medium (Oxoid, UK), respectively. The plates were incubated at 37 ℃ in a 5% CO 2 humidified jar for 7-14 days. These plates were monitored at least three times for growth of colonies (first time: after 3 or 4 days) until the end of the incubation period. The colonies with typical Legionella morphology were sub-cultured on BCYE agar with and without L-cysteine, blood agar and MacConkey agar plates. In case which the isolate could grow on BCYE agar with L. cysteine, but not on the other media, and stained as a gramnegative rod, it was considered as Legionella spp. [16]. In this study, L. pneumophila ATCC 33152 was used as a quality control.

DNA extraction
DNA extraction was performed using the boiling method. In this method, a few colonies were dissolved in Tris-EDTA (TE) buffer (10mM Tris, 0.1mM EDTA, pH 8.0). The suspension was incubated in a dry-block thermostat (Biosan, Latvia) at a temperature of 99 ℃ for 10 min and then, placed in an ice box at a temperature of −20 ℃ for 5 min. The suspension was subsequently centrifuged at 14000 rpm for 5 min and the supernatant was transferred to a new micro tube and kept at −20 ℃ for molecular assay [17]. The concentration and quality of DNA were evaluated by measuring the absorbance of A260 and A280 nm with a spectrophotometer (Thermo Fisher Scientific, USA) and agarose gel electrophoresis, respectively.

Gel electrophoresis and mip gene sequencing
The PCR products were loaded and separated onto a 2% agarose gel prepared in 1 X TAE (Tris/Acetate/EDTA) buffer. The amplicons were visualized using ultraviolet light gel documentation system (Protein Simple, San Jose, CA, USA) after staining with ethidium bromide 0.5 μg/mL (Cinnaclone, Tehran, Iran). A 100 bp DNA ladder was used as a size marker (Cinnaclone, Tehran, Iran). The amplified PCR products of mip gene for each isolate were purified with the Gene JET™ Gel Extraction Kit (Fermentas, Lithuania) according to manufacturer's instructions. The sequences of the products were determined by Bioneer Company, Korea using an ABI PRISM 7700 Sequence Detection System (Applied Biosystems, Foster City, Calif. USA) according to the standard protocol of the supplier. The primers used for sequencing were as follows: forward primer (Legmipfs), 5′-TTTATGAAGATGA(AG)A(CT)TGGTC(AG)CTGC-3′; and reverse primer (Legmip-r), 5′-TC(AG)TT(ATCG)GG(ATG)CC (ATG)AT(ATCG)GG(ATCG)CC(ATG)CC-3′.

Analysis of sequence data
The obtained mip gene sequences for each isolate were aligned separately and compared with all existing relevant sequences retrieved from GenBank database using the jPHYDIT program version 1. A percentage of similarity between the mip gene sequences of each isolate was determined by comparing sequences found to an in-house database of mip gene sequences (after data analysis by jPHYDIT program). The highest similarity percentage was considered as the identified species. For a confident identification, the similarity ≥98% to a sequence in the database was acceptable for submitted mip sequence.

Results
Overall, 20 Legionella spp. were detected by culture method in 144 water samples (13.9%) in Ahvaz city, southwest Iran. All isolates were confirmed as Legionella spp. by 16S rRNA gene PCR amplification. In this study, PCR of mip gene and its sequencing was used as reference identification method for detection of Legionella species (Figure.1). The water sources and distribution of identified Legionella species by mip sequencing are shown in Table 1. The result of mip sequencing and it`s analysis based on the hemology by jPHYDIT program revealed that L. pneumophila (with 13 isolates) was predominant (54.1%), whereas the Legionella species other than pneumophila, such as: L. worsleinsis, L. dumoffi, and L. fairfieldensis were accounted for 20.8%, 4.1% and 4.1%, respectively. In this study, the most common contaminated sources were hospital water baths (37 ℃) with 34.6% and tap water with 25%, respectively (Table 2). However, no Legionella spp were detected in the water samples of neonatal incubator, nebulizer in ICU, hospital dialysis device, dental unit water, air conditioner, and drink water cooler.   Table 2. Occurrence of Legionella spp in different water samples.

Discussion
Contamination of hospital water systems with Legionella spp. is a well-known cause of nosocomial Legionellosis. Inhalation of Legionella contaminated water aerosols could be cause of human infection. While the L. pneumophila is the main causative agent of legionellosis, the other species, such as L. micdadei, L. dumoffii, and L. bozemanii, are also known as other causes of disease in humans [18,19]. The culture method is still known as a gold standard for Legionella detection, but it needs to a long time for isolation of Legionella species. In this study, we attempted to investigate different water sources of Ahvaz city, southwest Iran for presence of Legionella spp by culture. In addition, we used the mip sequencing method for the differentiation of Legionella spp.
Phylogenetic studies of mip gene were gradually introduced for their greater capacity than the 16S rRNA in discriminating between of Legionella species [15,20]. In our study, some similarities which were observed in the mip gene amplicon among the 20 Legionella isolates, ranged from 98.57% to 100%. In this study, 13 isolated strains were identified as L. pneumophila (54.1%) from water samples by sequencing of mip gene. In consistence with our study, in a survey conducted on water systems in the southwest of Iran by culture and PCR methods, the prevalence of L. pneumophila was 41.1% [21]. In another study from Iran, 10 of 140 (7.1%) water samples were positive for Legionella species [22]. Our study confirmed the contamination of water sources of hospitals with the Legionella species that may play a key role as a risk factor for the patients' health in hospitals.
Moreover, it should be stressed that L. pneumophila is the most prevalent isolate in man-made aquatic environments, which should be seriously considered as a potential public health threat [23][24][25]. The past studies have been shown that the mip gene is a virulence factor which could provide the genetic evidence for high occurrence of L. pneumophila strains in man-made systems. This factor can resist to the intracellular killing against mammalian and protozoan phagocytic cells [26]. Legionella species, other than L. pneumophila also, may occur in man-made systems, however, more prevalence of L. pneumophila could be related to symbiotic interaction between this bacterium and free-living amoebae [27]. Furthermore, it seems that Legionella spp. other than pneumophlia with a lower infectivity and poor intracellular growth lead to more growth of virulent L. pneumophila strains in environmental sources [28]. The result of present study showed that L. worsleiensis (n = 5, 20.8%) was the second most observed isolate from the hospital water samples. Although the pathogenicity of L. worsleiensis is lower than L. pneumophila, however, a study has been claimed that presence of L. worsleiensis in environmental water samples (primarily in hospital water samples) might mask contamination with L. pneumophila, which could increase the infection risk with this organism [29]. The Legionella spp. could cause human pneumonia and accidentally induce other diseases, such as prosthetic valve endocarditis and septic arthritis [30,31].
Similar to some studies, L. fairfieldensis and L. dumoffi were isolated from our hospital water samples. Svarrer et al. showed that these isolates together with Legionella micdadei were the most common cause of the culture-verified Legionella non-pneumophila infections in Denmark [29].
In another study by Stølhaug et al. all of 12 non-L. pneumophila reference strains were identified with a high accuracy by mip gene sequencing. This study also indicated that the mip gene sequencing could differentiate Legionella species [32].

Conclusions
This study highlights the need of continuous monitoring and risk assessment of water supplies of hospitals in our region for Legionella spp. contamination. Furthermore, despite the fact that hospitals use a purified water system, 13.9% of the samples were positive for Legionella spp. Therefore, it can be concluded that the common methods of water purification and disinfection are not sufficient for purification of the water network from these bacteria and new policies should be put in place to control and eliminate them in water resources. Also, it is recommended that using the PCR method along with the mip gene sequencing could detect non-pneumophila Legionella species in routine surveillance of Legionella in water samples.

Limitation in this study
Although a neutralizing agent is needed in order to neutralize residual disinfectants, however no use of such agent in present study was identified as a limiting factor.