Molecular Identification of Free-Living Amoebae (Naegleria spp., Acanthamoeba spp. and Vermamoeba spp.) Isolated from Un-improved Hot Springs, Guilan Province, Northern Iran.

Background: This study was conducted to determine the presence and molecular identify of Acanthamoeba, Naegleria and Vermamoeba in unimproved hot springs. Methods: From Jul to Aug 2017, 54 water samples were collected from hot springs in different parts of the Guilan Province, North Iran. For the isolation of Acanthamoeba, Naegleria and Vermamoeba approximately 500 ml of the water samples were filtered through a cellulose nitrate membrane with a pore size of 0.45 μm. The filter was transferred onto non-nutrient agar plates seeded with Gram-negative bacteria (Escherichia coli) as a food source. The morphological key of page was used to identify free-living amoebae (FLA) using an inverted microscope, PCR amplification targeting specific genes for each genus and sequencing determined frequent species and genotypes base on NCBI database. Results: Fifteen of the 54 samples were positive by culture and/or PCR for Acanthamoeba and other FLA from unimproved hot springs. By sequencing the positive isolates, the strains were shown to belong to Acanthamoeba castellanii (12 case isolates belonged to T4 genotype), 4 cases of V. vermiformis, and 3 cases of N. australiensis, 2 cases of N. pagei and 1 cases of N. gruberi. Conclusion: Although FLA-mediated illnesses are not as high as in environmental distribution, but because of a poor prognosis, more investigations about FLA distribution in hot springs is critical. Hot spring may enhance exposure of the amoebae in individuals. Hence, more attention to unimproved hot springs is needed to prevent free-living amoebae mediated diseases.

Introduction ree-living amoebae (FLA) as amphizoic amoebae are a group of parasitic protozoa with the growth abilities in different natural environments such as soil and water. In appropriate conditions, they are pathogenic in animals and humans (1). FLA include many genera which cause serious diseases such as cutaneous ulcers, sight threatening keratitis and fatal encephalitis.
Acanthamoeba spp., Naegleria fowleri and Balamuthia mandrillaris are the most commonly reported causes in the world (2). Other genera in this group, including Sappinia diploidea, Vermamoeba and Vahlkampfia mix infection with other FLA could also lead to severe diseases with a lower incidence around the world (3)(4)(5). Acanthamoeba strains, especially ones that are potentially pathogenic, can tolerate extremes of temperature, osmolarity, and pH (6). Vahlkampfia and Naegleria genera are commonly found in warm freshwater.
N. australiensis and N. italica can cause infection in experimental animals but N. fowleri is able to infect animals and humans with possible life threatening contamination (7,8). Acanthamoeba spp. Vahlkammpfids and B. mandrillaris can cause serious infections in humans (1). In addition to Vermamoeba have as a suitable hosts for pathogenic microorganisms such as Legionella pneumophila (9,10). Other diseases related to FLA are Vermamoeba keratitis, and Vahlkampfia keratitis (3,5,11).
Hot springs are highly regarded for their therapeutic effects and how much of their use in Iran, both as a therapeutically use and as a tourist attraction, is growing; which will increase the chance of exposure to these amoebae (12)(13)(14). Guilan Province is a tourist areas of Iran which plays host to millions of Iranian and foreign travelers annually, and one of the tourist attractions of in this province is its hot springs (15). The presence of FLA in the hot springs of the provinces bordering Guilan has been confirmed (14,16). Parasitological meth-ods only recognize parasite contamination however, it is not possible to identify the exact species of parasite involved (17).
Hence, we aimed to survey the incidence of waterborne FLA belonging to Vermamoeba, Naegleria spp. and Acanthamoeba spp. isolated from unimproved therapeutic hot springs of Guilan Province using morphological and sequence based methods.

Geographical area of study
Guilan Provinces lies along the Caspian Sea. It has a plenty of annual rainfall, humid temperate weather and is known for its mild, moderate and Mediterranean-like climate (15) (Fig. 1).

Sampling
This cross sectional study (Code of ethics: IR.AJUMS.REC.1396.501) was performed from July to August 2017. To run this study, 54 water samples were collected from hot springs in different parts of the Guilan Province, North Iran ( Fig. 1). Six hot springs in Guilan, North of Iran were included in this study and nine samples per site (reservoirmargin-center) were obtained. Samples were collected from the surface of the waters (<5 cm below) (14).

Filtration, cultivation and cloning
Approximately 500 ml water samples were filtered using cellulose nitrate membranes with a pore size of 0.45 μm. Filters were transferred to non-nutrient agar (NNA) plates seeded with Escherichia coli as a food source (14,15). The morphological key of page was use for the identification FLA using an inverted microscope. Cloning of the suspected amoebae was performed using culture replicates method (11,18). The sampling points are indicated by blue circles ( )

DNA extraction, PCR analysis and Sequencing
DNA was extracted using GeNet Bio kit, according to manufacturer's instructions (GeNet Bio, South Korea). Four sets of primers were used in order to detect various FLA shown in Table 1. To carry out the PCR reaction, 25 ml of red master mix (Denmark) was combined with DNA (10 ng), 0.1 μM of each primer and distilled water. The cycling condition was set as pre-denaturation step for 3 min at 94 °C, followed by 35 repetitions at 94 °C for 35 sec, annealing steps were at 56 °C, 56 °C, 56 °C and 58 °C for 1 min (for Acanthamoeba, Vahlkampfiids, N. fowleri and Vermamoeba, respectively), and 72 °C for 1 min. All sequences were edited manually and analyzed with reference sequences by Chromas software program. The sequences were submitted to gene bank under following accession numbers: MH347242-MH347263.

Results
Of 54 water samples, 15 (27.7%) cases were positive for outgrowth of free-living amoebae. Indeed, the positive samples included 7 cases of Acanthamoeba, 2 cases of Vahlkammpfids, 1 case of Vermamoeba, 2 mixed cases of Acanthamoeba, Vahlkammpfids and Vermamoeba, 2 mixed cases of Acanthamoeba and Vahlkammpfids, and 1 mixed case of Acanthamoeba and Vermamoeba. Accordingly, Acanthamoeba were found in 12 (54.5%) samples as the most prevalent amoebae in the tested samples (Tables 1, 2).
For morphological determination of Acanthamoeba castellanii, the following characters were regarded; a double walled with a smooth wrinkled outer cyst wall and stellate endocyst (Figs. 2 and 3) for Vahlkammpfids round cysts with smooth wall and V. vermiforims round cysts with smooth wall but smaller Vahlkammpfids (Fig. 3 and 4). In electrophoresis of PCR products, twelve cases of Acanthamoeba demonstrated an approximately 500 bp band (Fig. 5). Sequencing analysis of 12 positive cases showed T4 (100%) genotypes with homology analysis of NCBI website revealing 95%-100% similarity.  Water PH and temperature were assessed in situ by using a portable pH meter (Digital tester , so that these parameters of hot springs were respectively measured as 23.4%-31.6 °C and 6.5-7.2 (Table 3). Moreover, sequencing analysis revealed 95%-100 % similarity with Vahlkammpfids and V. vermiformis. Accession numbers of nucleotide sequences were deposited in the GenBank database, and have been demonstrated in Table 3. Moreover, six hot springs, 83.4% (n=5), 66.6% (n=4) and 50% (n=3) were respectively positive for Acanthamoeba, Vahlkamfiids, and Vermamoeba amoebae.

Discussion
The present study is the first study on unimproved hot springs of Guilan Province, Northern Iran to determine the pathogenic free-living amoeba via molecular methods. In this investigation waterborne free living amoebae belonging to the Acanthamoeba T4 genotype, Naegleria (N. pagei, N. australiensis and N. gruberi) and Vermamoeba verformis were found in the unimproved hot springs of Guilan Province, Northern Iran and the present study is the second report of N. gruberi in the country. Acanthamoeba was detected in surface water of Guilan, previously (15).
No significant differences were shown between pH value (and temperature) and the presence/absence of Acanthamoeba, Naegleria, Vermamoeba. In previous studies, the T4 genotype was reported to be isolated from samples such as soil, hospital wards, surface waters, recreational water areas, dust sources and also hot springs in Iran (13,(19)(20)(21)(22)(23). In contrast with results reported by other studies that reported T15 and T3 as predominate genotypes in waters surveyed (11,24), the founding of PCR analysis and sequencing in the present work confirmed that the T4 genotype was the predominant type (Table 3).
Among the 47 known species of Naegleria, only N. fowleri has been reported to be pathogenic for human (8). An investigation on hot springs sources in Iran reported an increased occurrence of Naegleria genus in the tested samples (14). The pathogenic N. fowleri was not found in this study. To our knowledge, so far, there is no report on the presence of pathogenic N. fowleri in environmental sources of Iran. However, a clinical case of N. fowleri has been reported in the country (25).
In the present study, N. australiensis was the most prevalent species, which can be pathogenic to mouse (26). V. vermiforims was also one of the most detected FLA, based on molecular assays (Table 3). A case of Vermamoeba keratitis and a case of mix infection of V. vermiforims and Acanthamoeba were reported during previous studies (4,5,27).
To prevent infection and diseases related to free-living amoebae, hot springs should be periodically checked, in particular, during the summer season, when these surface water are used by thousands of tourists (28). The disease could originate to possess a seasonal mode of frequency in the region and serious monitoring for proper preparation against the disease should be in place (29).

Conclusion
Although FLA-mediated illnesses is not as high as in their environmental distribution, because of a poor diagnosis, more investigations about FLA distribution in hot springs is critical. Hot spring may enhance exposure of the amoebae to individuals. Hence, more attention to unimproved hot springs is needed to prevent free-living amoebae mediated diseases.