Isolation and identification of Acanthamoeba genotypes and Naegleria spp. from the water samples of public swimming pools in Qazvin, Iran

Free-living amoeba (FLA) are opportunistic protozoan parasites and some FLA, such as Acanthamoeba, Naegleria, Balamuthia and Sappinia are considered as pathogenic amoeba (Visvesvara et al. 2007; Henriquez & Khan 2009; Yousuf et al. 2013). The FLA can cause severe and even fatal diseases by invasion and causing damage to the central nervous system (CNS) and other organs. A wide range of diseases including amoebic encephalitis, keratitis and skin ulcerations have been reported in both immunocompetent and immune-deficient individuals (Khan 2006; Henriquez & Khan 2009). Moreover, the number of cases of amoebic keratitis (AK) in Iran has increased in the recent decade which is probably due to poor hygiene among contact lens users (Rezaeian et al. 2007). The amoeba can be the reservoir and vehicle of the microbial world, in particular pathogenic bacteria present in nature as they can transfer such microorganisms to humans, leading to enhanced amoeba infection (Rezaeian et al. 2008; Siddiqui & Khan 2012; Buse et al. 2016).

Amoebae have widespread distribution in different environments including water, soil, dust and air sources. Various water sources, such as pool, river and recreational waters can be contaminated with FLA. Thermal water was described as a suitable niche for Naegleria spp., therefore, water sources are considered as an important risk factor for amoebic disease (Visvesvara et al. 2007; Yousuf et al. 2013; Di Filippo et al. 2017; Xuan et al. 2017). It is obvious that swimming or bathing while wearing contact lenses leads to increased risk of AK.

Vegetative trophozoites and resistant cysts of FLA are widely distributed in nature. In harsh conditions, amoeba can be enclosed by a double-walled cyst structure. The cysts of amoeba withstand different disinfectant solutions, unpleasant environmental conditions such as severe dryness, extremes in temperatures, pH and improper osmolarity (Khan 2006).

Up to now, 20 genotypes (T1–T20) of Acanthamoeba have been found in environmental and clinical specimens, with T4 genotype as the most common genotype that demonstrates higher pathogenicity among patients (Khan 2006; Nuprasert et al. 2010; Corsaro et al. 2015). To date, several genotypes including T2, T3, T4, T5, T9, T11 and T13 have been reported from different parts of Iran (Nazar et al. 2011; Rahdar et al. 2012; Solgi et al. 2012; Behniafar et al. 2015; Hajialilo et al. 2016). There are various species of Naegleria such as N. fowleri, N. australiensis, N. lovaniensis, N. italica, etc., but among those only N. fowleri is a true pathogen capable of causing primary amoebic meningoencephalitis (PAM) in individuals. In addition, N. lovaniensis is the only non-pathogenic thermophilic species of Naegleria, with a close evolutionary relationship to N. fowleri (Carter 1968; Di Filippo et al. 2017). Several species of Naegleria including N. fowleri, N. australiensis, N. pagei, N. gruberi, N. americana, N. dobsoni, N. clarki and N. fultoni have been found in different sources of water and among patients in Iran (Movahedi et al. 2012; Niyyati et al. 2012, 2015; Javanmard et al. 2017; Latifi et al. 2017).

Numerous studies have been conducted on different sources of water such as tap water, hot and mineral spring waters, however, few researches were focused on swimming pool water with Jacuzzi tubs. Since no previous study was based on such water sources, the possible presence and distribution of FLA in public swimming pool water sources of Qazvin province, the current study aimed to identify and genotype the FLA isolated from swimming pool water sources and Jacuzzi tubs within Qazvin province.

This cross-sectional study was carried out from December 2016 to April 2017. In total, 166 water samples were collected from swimming pools and Jacuzzi tubs in Qazvin province. The province is located in the northern margin of central Iran. Water samples were collected from the surface and floor of target swimming pools, hot and cold tubs several times. At the time of study, there were 17 public swimming pools, and among those, 12 voluntarily participated in the study. A total of ten samples were randomly collected from each swimming pool (including five surface water samples and five deep water samples). One water specimen was also taken from hot and cold tubs, separately. The water samples were gathered into 500 mL sterile flasks, and then transferred to the Department of Medical Parasitology and Mycology at the School of Medicine, Qazvin University of Medical Sciences, Iran. Calcium hypochlorite was used on a daily basis for disinfecting the swimming pools.

Water specimens were filtered through nitrocellulose membrane filters (0.45 μm pore size). The membranes were cultured on 1.5% non-nutrient agar (NNA) plates, seeded with heated inactivated suspension of Escherichia coli bacteria. The plates were incubated at 30 °C for up to 15 to 30 days to grow amoeba, followed by daily examination of the cultures under light microscope to observe the presence of trophozoites and cysts of amoeba (Solgi et al. 2012). The positive plates were cloned several times to eliminate bacterial and fungal contamination (Lorenzo-Morales et al. 2005; Behnia et al. 2017).

Pathogenicity tests' positive plates of Acanthamoebae were selected for osmo-tolerance and thermo-tolerance assays. Bactoagar medium with two concentrations of mannitol (0.5 M and 1 M) were used for osmo-tolerance assay while thermo-tolerance assay evaluated the growth ability of Acanthamoebae at two different temperatures of 37 and 42 °C. All plates were monitored daily for a week. Controls were applied to prevent likely errors (Khan 2001; Hajialilo et al. 2016).

Trophozoites and cysts of amoebae were collected from the plates using phosphate buffered saline (PBS), at a pH of 7.2, followed by treating the FLA with lysozyme and glass beads. High pure polymerase chain reaction (PCR) Template Preparation Kit (Roche, Mannheim, Germany) was used for DNA extraction. PCR reaction was set up with specific primers for Acanthamoeba, JDP1 5′-GGCCCAGATCGTTTACCGTGAA-3′and JDP2 5′-TCTCACAAGCTGCTAGGGAGTCA-3′ to amplify the 500 bp length fragment within the 18S rRNA gene region.

The primers used for Naegleria were designed by Beacon Designer7 and Primer-BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) and included NA1 5′-AACCTGCGTAGGGATCAT-3′ and NA2 5′-TTTTCTTTTCCTCCCCTTAT-3′ which amplified an approximately 400 bp piece. PCR reaction for both Acanthamoeba and Naegleria were performed in a total volume of 30 μL containing a ready-made mixture of Amplicon (Taq DNA Polymerase Master Mix RED, Denmark), template DNA, 0.1 μM of each primer along with distilled water. The cycling conditions for Acanthamoeba were adjusted according to Lasjerdi et al. (2011), with some modifications as follows: initial denaturing phase of 94 °C for 4 min, followed by 35 cycles at 94 °C for 30 s, annealing at 64 °C for 45 s and 72 °C for 45 s, and a final extension step at 72 °C for 7 min (Lasjerdi et al. 2011). The thermal cycling conditions used for Naegleria were initial denaturing phase at 95 °C for 5 min, followed by 35 cycles at 95 °C for 20 s, annealing phase at 57 °C for 20 s and at 72 °C for 30 s, and a final extension step at 72 °C for 5 min. Electrophoresis and staining with ethidium bromide were performed to visualize the specific band under UV light.

Purification and sequencing of PCR products were conducted using an automatic ABI3130 sequencer machine (Applied Biosystems, USA). The DNA sequences were edited by Chromas (Version 1.0.0.1), and compared to BLAST GenBank database against eukaryotic sequences. The sequences obtained in our experiments were deposited in the GenBank database under the Accession numbers Acanthamoeba, Protacanthamoeba and Naegleria: MH024474-MH024493, MH304644 and MH304645. The phylogenetic tree was constructed using the MEGA7 software; maximum-likelihood algorithm with Tamura-3 parameter substitution model was applied. Bootstrap analysis was performed based on 1,000 replications. Balamuthia mandrillaris sequence was used in the dendrogram as an outgroup (Figure 1) (Table 1).

In total, 18.6% (31/166) of water samples collected from the swimming pools were positive for FLA. While both Acanthamoeba and Protacanthamoeba were found to have contaminated the surface and deep water specimens, no Naegleria was detected in water samples of the swimming pools. Out of 14 water specimens collected from hot Jacuzzis, 14% (2/14) were contaminated with Naegleria spp. while no amoeba was found in cold Jacuzzis. Double-walled cysts, based on irregular inner layer of cyst (Acanthamoeba) and round shape cyst of Naegleria are shown in Figure 2. Positive specimens were examined by PCR and showed specific bands of Acanthamoeba and Naegleria. All 22 culture positive specimens showed bands on agarose gel. According to molecular analysis, 63.6% (14/22), 27.3% (6/22) and 9% (2/22) samples were Acanthamoeba (T3, T4 and T11 genotypes, Acanthamoeba spp.), P. bohemica and Naegleria species, respectively. The sequences detected for both N. lovaniensis isolates QIN140 (MH304644) and QIN163 (MH304645) demonstrated 100% homology to the reference genes in the GenBank database. According to the results obtained for pathogenicity assays, 35% (7/20) of the Acanthamoeba and Protacanthamoeba isolates revealed high pathogenicity, among those 50% (4/8) belonged to T3 genotype, 25% (1/4) to T4 genotype and 33% (2/6) related to P. bohemica. Other isolates of Acanthamoeba were found to be of low pathogenicity (Table 2). The P. bohemica species of the present study were in a cluster with other species of the Protacanthamoeba and reference sequence of Protacanthamoeba bohemica, therefore N. lovaniensis species were in a separate cluster along with the reference sequence of Naegleria lovaniensis (Figure 1) (Table 1).

The results of this study demonstrated Acanthamoeba T3, T4 and T11 genotypes, as well as Protacanthamoeba species in the public swimming pools of Qazvin province. T3 genotype was identified as the dominant genotype of this protozoan parasite in the area. Comparison of molecular studies of FLA in different countries shows geographical variation among the species and genotypes of FLA specimens collected from public swimming pools in different regions of the world. For instance, six species of Acanthamoeba including A. polyphaga, A. mauritaniensis, A. castellanii, A. royreba, A. triangularis and A. rhysodes (Al-Herrawy et al. 2014) and three genotypes of Acanthamoeba (T3, T4 and T5 genotypes) have been reported from Egypt and Brazil (Caumo & Rott 2011), respectively. Acanthamoeba spp. and Naegleria spp. were also reported in swimming pools in Malaysia (Init et al. 2010). A survey on heated indoor swimming pools in Switzerland detected A. lenticulata (Gianinazzi et al. 2009a). Genotype diversity is also observed among FLA isolated from different parts of the country. Investigation on fixed and floating biofilms of swimming pools and hot tubs in the city of Tabriz (Iran) found Acanthamoeba T3 and T4 genotypes (Poor et al. 2018). In another study reported from Semnan province, in the northern half of Iran, the authors showed the presence of Vermamoeba vermiformis in water samples of swimming pools (Javanmard et al. 2017). The authors of a study carried out on water samples of swimming pools in the city of Shiraz claimed the presence of Acanthamoeba T4 and T5 genotypes as well as V. vermiformis (Armand et al. 2016). Acanthamoeba T3, T4 and T5 genotypes were also reported in the water samples collected from pools and ponds in southeast Iran (Aghajani et al. 2016). Rezaeian et al. (2007) isolated Acanthamoeba spp. from water specimens taken from swimming pools in Tehran. Although T4 genotype of Acanthamoeba is the most abundant and with higher pathogenicity, compared to other genotypes of amoeba found among Acanthamoeba keratitis patients and environmental sources (Niyyati et al. 2009; Nuprasert et al. 2010; Corsaro et al. 2015), our present research revealed the predominance of T3 genotype as the most common genotype among the other isolates with different genotypes. Aligned with our survey, a study on water sources in Osaka, Japan showed T3 genotype of Acanthamoeba as the dominant type among the isolates (Edagawa et al. 2009).

In the current study we found P. bohemica among the specimens. Protacanthamoeba is classified in the order amoebida and a member of the family Acanthamoebaidae (Fuerst et al. 2015). P. bohemica has been introduced as the species of Protacanthamoeba (Dyková et al. 2005). The species was reported in samples obtained from water sources in Switzerland (Gianinazzi et al. 2009b). Neither of these two amoebae were detected in humans. Four isolates (QIA42, QIA48, QIA85 and QIA164) of Acanthamoeba that belong to the T3 genotype were demonstrated to be highly pathogenic, compared to the QIA97 isolate as the sole T4 genotype which was pathogenic in osmic-thermotolerance pathogenicity assay. The results of pathogenicity tests clarified that T3 genotype can be pathogenic to humans, although more research including in vivo experiments is needed to confirm the exact pathogenicity of various genotypes of amoeba. The present research found no Acanthamoeba in hot Jacuzzi water samples, whereas in a study reported from Brazil the presence of Acanthamoeba was reported in water samples of hot tubs (Fabres et al. 2016). As expected, the only Naegleria identified was isolated from a hot Jacuzzi with no FLA in cold Jacuzzi, indicating that a cold Jacuzzi is an inappropriate environment for the growth of FLA. This study showed that there is no tropism in the distribution of FLA in both surface and deep water samples of swimming pools.

The species of the genus Naegleria can cause fatal central nervous system infections (De Jonckheere 2014). A case of primary amoebic meningoencephalitis with N. fowleri infection in a five-month-old male infant was found in Iran (Movahedi et al. 2012). Naegleria is considered as a thermophilic amoebae with hot springs or warm waters as the suitable habitat for this amoebae (De Jonckheere 2014). The pond water of parks in Mashhad city were contaminated with some Naegleria spp. (Najafpoor et al. 2018). In the present study, Naegleria (N. lovaniensis) was found in hot tubs of swimming pools whereas no contamination was identified in cold tubs, implying that low temperature is an unsuitable condition for growth of FLA. Our study is the first report of N. lovaniensis in the country. Although several species of Naegleria have been identified (Di Filippo et al. 2017), it is only N. fowleri that is proven to be pathogenic for humans. Currently, the non-pathogenic N. lovaniensis is considered as an amoeba with close evolutionary relationship to N. fowleri (Di Filippo et al. 2017).

Swimming in contaminated water while wearing contact lenses is an important risk factor of AK (Khan 2006) and an increase in the rate of AK across the country could be due to improper cleaning of swimming pool water which is a major public health concern (Rezaeian et al. 2007). The standard concentration of free chlorine is around 1–3 ppm (Rasti et al. 2012) and both swimming pools and hot and cold tubs in our study were found to be within the standard range of chlorine concentration. Our study demonstrated that the recommended concentration of free chlorine failed to prevent the growth of FLA. In conclusion, T3 and T4 genotypes were found to be the most common genotypes of Acanthamoeba in the swimming pools of the study area with the potential to threaten the health of individuals who swim in these pools. The genotypes and species of the amoeba may have pathogenic potential and need re-evaluation, so this study highlights the necessity for taking strict measures over the sanitation of recreational water sources, specifically those of swimming pools, to protect individuals against FLA infection.

This research was financially funded by the Cellular and Molecular Research Center, Qazvin University of Medical Sciences, Qazvin, Iran (Grant No: 850). The authors of the present study would like to sincerely thank the Health Center of Qazvin province and also the managers of the swimming pools in the study area for their kind assistance in taking water samples from the pools, and lastly the Department of Parasitology and Mycology at Medical School, affiliated to Qazvin University of Medical Sciences for their technical assistance. We also thank Dr. Ali-Asghar Pahlevan for editing the final version of the English manuscript. The results described in this article are part of an MSc dissertation conducted by Mrs. Nastaran Paknejad at the Department of Medical Parasitology & Mycology. The procedure of this project was approved by the Research Ethics Committee of Qazvin University of Medical Sciences (Code no: IR.QUMS.REC.1396.262).