High occurrence of Acanthamoeba spp. in the water samples of public swimming pools from Kerman Province, Iran

Acanthamoeba spp. is a free-living amoeba that can cause major infections in humans, including keratitis and granulomatous encephalitis. Thus, water resources play an important role in transmitting Acanthamoeba spp. infection to humans. The purpose of this study was to inves-tigate the presence of Acanthamoeba spp. in public swimming pools from three cities of Kerman Province, southeastern Iran. Eighty water samples of 20 public indoor swimming pools were taken from Kerman, Jiroft, and Kahnauj cities. Water temperature (°C), pH, and free chlorine concentration (ppm) were measured. Filtration and cultivation were applied on non-nutrient agar medium. The polymerase chain reaction was applied by using the genus-speci ﬁ c primers (JDP1 and JDP2) on positive samples; these primers can amplify the 423 – 551 bp fragment. Eighteen of the 20 swimming pools (including 32/80; 40% samples) were contaminated with Acanthamoeba spp. All swimming pools of Jiroft and Kahnauj and 88.2% of swimming pools in Kerman were contaminated. As such, all 32 Acanthamoeba isolates were ampli ﬁ ed using the JDP primer pairs. Two genotypes, T3 and T4, were also identi ﬁ ed. The present research is the ﬁ rst to report Acanthamoeba spp. in public swimming pools from Kerman Province. Due to high occurrence of this protozoan, it is recommended to use warning signs around swimming pools to create awareness of this infection.


INTRODUCTION
Free-living amoebae from the genus Acanthamoeba have a broad global distribution and can be found in a wide range of environmental sources such as water, soil, and air (Trabelsi et al. 2012;Niyyati & Rezaeian 2015). Acanthamoeba spp. has a biphasic life cycle that exhibits two distinct forms: a vegetative form (trophozoite) and a resistant form (cyst) (Paknejad et al. 2020;de Lacerda & Lira 2021). In favorable conditions, trophozoite forms are identified by having spine-like structures (known as acanthopodia) (Khan 2001). In this stage, trophozoites are able to replicate, move, feed, or adhere to the host cell surface through acanthopodia (Khan 2003;de Lacerda & Lira 2021). During the unfavorable condition, Acanthamoeba spp. can be surrounded by a resistant double-walled cyst structure (Gabriel & Panaligan 2020). These cysts withstand adverse environmental conditions such as very high or low temperatures, severe dryness, ultraviolet (UV) radiation, the presence of chemical compounds (i.e., accumulation of metabolic products and toxins), disinfectant solutions, changes in osmolarity, and pH (Aksozek et al. 2002;Dudley et al. 2005;Lonnen et al. 2010;Taher et al. 2018).
According to published studies, most reports of AK occur after water exposure or a history of swimming in lakes and pools while wearing soft contact lenses (Ibrahim et al. 2007;Lindsay et al. 2007). Therefore, these environmental sources can be considered as major potential risks for human infection. Since there was no information regarding the status of Acanthamoeba spp. in Kerman Province in the southeastern region of Iran, the main aim of the present study was to investigate the presence of Acanthamoeba using morphological and molecular tools in public swimming pools from three cities of Kerman Province (Kerman, Jiroft, and Kahnauj).

Sampling
During June-August 2018, a total of 80 water samples (∼500 mL for each sample) were collected into sterile bottles from 20 public indoor swimming pools of three cities of Kerman Province in the southeastern region of Iran ( Figure 1). These cities were Kerman (17 swimming pools including 68 samples), Jiroft (two swimming pools including eight samples), and Kahnuj (one swimming pool including four samples) ( Table 1). Two sampling stations, 1 m from the pool wall and the middle of the pool, were selected, as listed in Table 1. Moreover, the water temperature (°C), pH, and free chlorine concentration (ppm) were measured during sampling. Then, all of the samples were immediately transported to the Laboratory of Parasitology and Mycology, Department of Kerman University of Medical Sciences, Iran.

Filtration, cultivation, and microscopy detection
At first, the samples were thoroughly stirred to mix the contents. After shaking, each water sample was filtered through an autoclaved 2.5 μm pore filter size (Whatman, Grade 42, UK) by mild suction using a vacuum or pressure pump (a new sterile membrane was used for each water sample) (Solgi et al. 2012). Then, the filters were transferred onto 1.2% non-nutrient agar (NNA) medium covered with autoclaved Escherichia coli K12 (HB101), a noninvasive laboratory strain (Tanveer et al. 2013). All plates were sealed with parafilm and the three plates per sample were incubated at 30, 37, or 42°C for up to 30 days (Solgi et al. 2012). The subculture was performed for all the positive plates on fresh NNA plates to obtain pure and noncontaminated isolates (Solgi et al. 2012). Finally, the microscopic detection of Acanthamoeba was conducted based on Page keys (Page 1988) using inverted microscopy.

DNA extraction and polymerase chain reaction amplification
Trophozoites and cysts were harvested from plates and washed using sterile phosphate-buffered saline (PBS, pH 7.4). Next, the extraction of DNA was performed using the DNAeasy kit (Qiagen, Basel, Switzerland) according to the manufacturer's instructions.
DNA was amplified by using the genus-specific primers (JDP1 and JDP2) for 18 s rRNA of Acanthamoeba spp. (Schroeder et al. 2001;Behniafar et al. 2015), including the primer pair JDP1 (5 0 -GGCCCAGATCGTTTACCGTGAA-3 0 ) as forward primer and JDP2 (5 0 -TCTCACAAGCTGCTAGGGAGTCA-3 0 ) as reverse primer (Schroeder et al. 2001). These primers can amplify the 423-551 bp fragment. Then, a total of 20 μL volume of polymerase chain reaction (PCR) were fixed by using 10 μL (2Â) of Taq Master Mix (Cinnagen, Iran), 25 pmol of each primer, distilled water, and 2 μL of DNA template. The PCR was carried out under the following conditions: 45 cycles of denaturation at 95°C for 1 min, followed by 45 repetition cycles at 95°C for 1 min, annealing at 60°C for 1 min and 72°C for 2 min, and final extension at 72°C for 5 min. Finally, PCR Uncorrected Proof products were electrophoresed by using 1.5% agarose gel stained with an ethidium bromide solution and visualized under UV light.

Sequencing and genotype identification
In this stage, PCR products (randomly) were prepared for sequencing using a sequencer machine (Applied Biosystems ® 3130X Automatic Genetic Analyzer) followed by performing homology analysis using the Basic Local Alignment Search Tool (BLAST) program to compare the identified sequence with those available in the GenBank sequence database. Table 1, a high abundance of potentially pathogenic Acanthamoeba spp. was detected in public swimming pools included in this research. Accordingly, 18 of the 20 swimming pools (including 32/80; 40% positive samples) were

Kerman 1
In the middle of the pool (2) 8 1.5 31 1 One meter away from the pool wall (2) 7.4 0.5 28 0 2 In the middle of the pool (2) 7.8 1.5 32 0 One meter away from the pool wall (2) 7.7 2.5 31 1 3 In the middle of the pool (2) 7.8 0.5 32 1 One meter away from the pool wall (2) 7.4 0.5 35 0 4 In the middle of the pool (2) 7.8 1 32 0 One meter away from the pool wall (2) 7.6 1.5 32 0 5 In the middle of the pool (2) 7.6 2.5 31 1 One meter away from the pool wall (2) 7.8 1.5 30 1 6 In the middle of the pool (2) 7.4 2 31 1 One meter away from the pool wall (2) 7.2 1 33 0 7 In the middle of the pool (2) 7.8 1.5 27.5 1 One meter away from the pool wall (2) 7.8 2 32 1 8 In the middle of the pool (2) 7.8 1 34 0 One meter away from the pool wall (2) 7.5 1.5 31 1 9 In the middle of the pool (2) 8.2 2.5 31 1 One meter away from the pool wall (2) 7.6 1 28 1 10 In the middle of the pool (2) 7.8 2.5 27 0 One meter away from the pool wall (2) 7.5 1.5 33 2 11 In the middle of the pool (2) 8.2 2.5 32 1 One meter away from the pool wall (2) 7.8 3 30 1 12 In the middle of the pool (2) 8 1 30 1 One meter away from the pool wall (2) 7.8 2 30 0 13 In the middle of the pool (2) 7.8 2 33 0 One meter away from the pool wall (2) 7.5 1 32 2 14 In the middle of the pool (2) 7.8 2 30 1 One meter away from the pool wall (2) 7.8 2.5 30 0 15 In the middle of the pool (2) 7.6 1.5 32 2 One meter away from the pool wall (2) 7.8 0.8 31.5 1 16 In the middle of the pool (2) 8 3 30 1 One meter away from the pool wall (2) 7.8 1 32 0 17 In the middle of the pool (2) 7.6 1.5 32 0 One meter away from the pool wall (2) 7.5 1.5 28 0 Jiroft 18 In the middle of the pool (2) 7.8 2 22 2 One meter away from the pool wall (2) 7.6 2 27 2 19 In the middle of the pool (2) 7.6 1 26 2 One meter away from the pool wall (2) (Page 1988), Acanthamoeba genus was identified by the presence of double-walled cysts with various shapes of endocysts, such as stars and triangles (inner wall often polygonal or stellate and outer wall often rippled or wrinkled with an approximate diameter of 15-20 μm), and flat trophozoites with acanthopodia (finger-like tapering pseudopodia) (Figure 2). According to the sampling station, 42.5% (17/40 samples) and 37.5% (15/40 samples) of infections were detected in the middle and within 1 m away from the pool wall, respectively (Table 1). Moreover, the frequency of Acanthamoeba spp. is listed in Table 1 based on temperature, pH, and chlorine concentration. All 32 Acanthamoeba isolates were amplified the 423-551 bp product using the JDP primer pairs. According to the reference (Schroeder et al. 2001), Acanthamoeba species can be identified based on band size (Table 2). In this regard, 22 out of 32 samples (68.75%) were amplified in the band size of 450 bp, and the rest were amplified in the sizes ranging between 500 bp (18.75%) and 550 bp (12.5%) ( Table 2). Some of the amplified samples are shown in Figure 3. Three random samples (one sample from each band size) were sequenced, of which two isolates belonged to T3 (A. griffini) and one isolate belonged to T4 (A. hatchetti). As such, all three samples were submitted in the GeneBank under the accession numbers MT292605, MT292606, and MT292607.

DISCUSSION
Although there are numerous reports of Acanthamoeba spp. in swimming pool water samples by various researchers around the world (Magnet et al. 2012;Mahmoudi et al. 2012), only sporadic studies have been performed on Acanthamoeba spp. in different parts of Iran. Recently, a meta-analysis study on the prevalence of Acanthamoeba spp. was published in Iran (Motavallihaghi et al. 2019), which showed that no studies have been conducted in Kerman Province so far. Therefore, providing results from the status of this free-living amoeba in Kerman Province can be important for policymakers and health officials in order to control and prevent this protozoan. The current study has highlighted that all swimming pools in Jiroft and Kahnauj cities and 88.2% of swimming pool water sources in Kerman city have been contaminated by Acanthamoeba spp. This prevalence rate is almost congruent with reports from previous studies in the river water samples of Guilan (88%) (Mahmoudi et al. 2015) and reports on surface water samples of Shiraz (99.6%) (Ghalehbin et al. 2014) in Iran. Moreover, the present contamination rate was higher than results obtained from river waters of Tonekabon (23%)   (Behravan et al. 2015) in Iran. In comparison with other countries, the contamination rate of Acanthamoeba spp. stated in the present study was higher than tap water from Brazil (9.5%) (Winck et al. 2011), river water samples from Turkey (44%) (Koyun et al. 2020), and environmental water samples from China (14.68%) (Lass et al. 2017). Therefore, all of these studies indicate that Acanthamoeba spp. is found in a broad range of water sources.
In the present study, the high prevalence of Acanthamoeba spp. could be a serious health risk, especially for high-risk groups (e.g., immunocompromised individuals and those who wear contact lenses) in this area. Therefore, awareness of high-risk individuals regarding the avoidance of contact with untreated and contaminated water, as well as swimming in public pool water while wearing contact lenses, could prevent Acanthamoeba-related infections such as AK and GAE (Niyyati et al. 2014;Poor et al. 2018). In addition, as shown by the results of molecular examinations and sequencing, the presence of major species and genotypes (T3 and T4) greatly increases the importance of attention and awareness of this microorganism. As such, one of the most important reasons for controlling Acanthamoeba spp. in different water samples is that this parasite can be used as a substrate for the survival, growth, and transmission of bacteria (i.e., Legionella, Mycobacteria, and Vibrio species) (Denet et al. 2017;Muchesa et al. 2017); thus, the co-existence of Acanthamoeba spp. with opportunistic bacteria may pose a potential health risk to immunocompromised individuals.
Although we used a specific primer to determine the different species of Acanthamoeba spp., it is recommended to use the sequencing technique due to the precise determination of genotypes. In this study, due to limited financial resources, only three positive samples were sequenced.

CONCLUSION
Overall, the present study reveals a significant occurrence of potentially pathogenic Acanthamoeba spp. in the water of the public swimming pools from Kerman Province. The use of improved filtration methods in various water sources by filters with smaller pores could be of greater importance in order to prevent water contamination. Moreover, it is recommended to use warning signs around swimming pools to make people aware of this infection.