Isolation and molecular identification of free-living amoebae (Naegleria spp., Acanthamoeba spp. and Vermamoeba spp.) from mineral springs in Guilan Province, northern Iran

Free-living amoebae (FLA) include many genera which cause serious diseases such as sight-threatening keratitis, cutaneous ulcers and fatal encephalitis. Acanthamoeba spp., Naegleria fowleri and Balamuthia mandrillaris are the most commonly reported causes in the world (Hooshyar et al. 2013; Javanmard et al. 2017; Feiz-Haddad et al. 2019a). Other genera in this group, including Sappinia, Vermamoeba and Vahlkampfia, are causative agents of disease with a lower incidence around the world (Abedkhojasteh et al. 2015; Javanmard et al. 2017). Acanthamoeba is widely distributed in a variety of environments including ocean sediments, tap water, ponds, hydrotherapy pools, lakes and hot springs (Feiz-Haddad et al. 2019b). There have been several reports of finding the source of Acanthamoeba (belonged to T4, T5, T7 and T11 genotype), Naegleria gruberi and Vahlkampfia vahlkampfia from mineral water in Brazil and Mexico (Rivera et al. 1981; Maschio et al. 2015). Further, Todd et al. (2015) reported Acanthamoeba from 50.6 to 17.3% recreational (mineral springs, ponds, lagoons, rivers, beaches and streams) and domestic water, respectively, from Jamaica. Free-living amoebae such as Acanthamoeba spp. can act as a Trojan horse and reservoir for pathogenic microorganisms such as Protozoa, bacteria, viruses and yeasts (Khan 2009).

To date, the Acanthamoeba genus, based on 18S rDNA sequencing, is classified into 21 genotypes (T1–T21) (Corsaro et al. 2017).

At present, in Iran Acanthamoeba genotypes related to keratitis are T4, T3, T2, T11, T13 and T15 (Niyyati & Rezaeian 2015). Acanthamoeba keratitis (AK) is mostly seen among healthy individuals and the young, and most of them (80%) have a history of wearing contact lenses (CLs), and it is seen in people who never use CLs as well (Feiz-Haddad et al. 2019b). In a study carried out by Shokri et al. (2016) in northern Iran on water sources, out of 77 water samples taken from different water sources within the Mazandaran Province in northern Iran (Sari city and suburbs), 83.3% of sequenced isolates belonged to the T4 genotype and the rest belonged to the T2 genotype (Shokri et al. 2016). The presence of FLA in the recreational water of the provinces bordering Guilan has been confirmed (Latifi et al. 2016, 2017; Feiz-Haddad et al. 2019a). Also, there are some studies indicating the presence of Acanthamoeba in river water samples in Guilan Province, northern Iran (Mahmoudi et al. 2012, 2015a, 2015b). However, there is no information regarding free living amoeba in mineral springs in these areas. Guilan Province has numerous mineral springs in different cities. This study was conducted due to the lack of research regarding the genotypes Acanthamoeba, Naegleria and Vermamoeba in mineral springs of Guilan Province in northern Iran.

Five mineral springs in Guilan Province, northern Iran, were included in the current study and five samples per site were obtained (Latifi et al. 2017). Rudbar, Fuman, Amlash, Rasht and Rudsar (Figure 1) are five cities in this province known for their recreational and therapeutic mineral springs and these are included in the present study (Latifi et al. 2017). Briefly, a volume of 500 mL of surface water was collected from mineral springs and transferred within 4 hours to the laboratory to be processed (Latifi et al. 2017). Physical parameters such as the pH and temperature of water were measured by a portable pH meter (Digital tester DMT-20) (Dodangeh et al. 2018).

Samples were filtered using cellulose nitrate membranes with a pore size of 0.45 μm as reported in a previous study (Latifi et al. 2017). The filters were then inverted and cultured onto 2% non-Nutrient agar plates covered with a layer of Escherichia coli. Incubation of samples was carried out at 30 °C in order to detect Acanthamoeba spp. Naegleria spp. Vermamoeba and 45 °C in order to detect N. fowleri. The morphological key of Page (Page 1988) was used for the identification FLA using both an inverted microscope and wet-mount method. Cloning of the suspected amoebae was performed using the culture replicates method as previously described (Latifi et al. 2017).

In this study, cloned amoebae in plates were harvested using phosphate buffered saline (pH: 7.2) and the amoebae were precipitated by centrifuging for 5 min at 2,000 rpm. DNA was extracted using the modified phenol-chloroform method (Mahmoudi et al. 2012; Javanmard et al. 2017) and GeNet Bio kit, according to the manufacturer's instructions (GeNet Bio, South Korea). To carry out the polymerase chain reaction (PCR), 25 μL Ampliqone (Taq DNA Polymerase Master Mix RED, Denmark) was combined with DNA (10 ng), 0.1 μM of each primer and distilled water. Four sets of primers were used in order to detect various FLA which are shown in Table 1. In the present study, we used two primer sets, the Naegleria genus-specific and N. fowleri species-specific, designed from the ITS1-ITS2 region by Pelandakis et al. (2000), Latifi et al. (2017) and Javanmard et al. (2017) who had previously provided successful results. The ribosomal ITS sequence was reported to be a powerful tool for detecting inter- and intra-species differences of various organisms, including Vahlkampfia species (Garstecki et al. 2005) and Naegleria (De Jonckheere 1998). As in Naegleria, the differences between species (intra-species of N. fowleri) and genus (inter-species) were due to the polymorphism sequence that occurred at the ITS1 and ITS2 regions, respectively (Johan 2002). The ITS region was also being used for the identification of the new Naegleria isolates (Sheehan et al. 2003; Ithoi et al. 2011). The cycling condition was set as pre-denaturation step for 3 min at 94 °C, followed by 35 repetitions at 94 °C for 35 s, annealing steps were at 56, 56, 56 and 58 °C for 1 min (for Acanthamoeba, Vahlkampfiids, Naegleria fowleri and Vermamoeba, respectively), and 72 °C for 1 min (Javanmard et al. 2017). The expected sizes of PCR amplicons were revealed by electrophoresis on 1.5% horizontal agarose gel in Trisborate-EDTA buffer and stained with a solution of Safe Stain.

PCR products were submitted to DNA sequencing using an ABI 3130XL automatic sequencer. Blast analysis of the sequences with others available in the GeneBank data was performed using BLAST software from the National Center for Biotechnology Information (NCBI) site. The highest homology was the base of species identification (Latifi et al. 2017). The sequences were submitted to the gene bank under the following accession numbers: MK422924, MK441743, MK441744, MK441745-MK441748, MK441750 and MK441752-MK441754.

The pH and temperature of the mineral springs are shown in Table 2. Of the 25 water samples collected from mineral springs in northern Iran, 11 (44%) were positive for Acanthamoeba spp., Naegleria spp. and Vermamoeba spp. based on the morphological criteria of Page (1988). The Acanthamoeba cysts were characterized as being double walled; the ectocyst and endocyst (Figure 2). For Vahlkampfiids round cysts with a smooth wall and Vermamoeba vermiformis round cysts with a smooth wall but smaller Vahlkampfiids refer to Figures 3 and 4 respectively. These 11 isolates were cloned successfully (Figures 2–4). Positive cultures were analyzed by PCR and genotypes base on the NCBI database. In electrophoresis of PCR products, Acanthamoeba demonstrated an approximately 500 bp band (Figure 5). Acanthamoeba spp. was detected in three of the extracted DNA by PCR, using the JDP primer pairs, which are specific for the Acanthamoeba genus. Water pH and temperature were assessed in situ by using a portable pH meter (Digital tester DMT-20), so that these parameters of Mineral springs were respectively measured as 16–27 °C and 6.1–7.2 pH (Table 2). By sequencing the positive isolates, the strains were shown to belong to Acanthamoeba castellanii (three case isolates belonged to T4 genotype), three cases of Vermamoeba vermiformis and two cases of N. australiensis, two cases of N. pagei and one case of N. gruberi (Table 3).

The present study is the first study on the mineral springs of Guilan Province, northern Iran, to determine the pathogenic free-living amoeba via molecular methods. This study reports the presence of potentially pathogenic Acanthamoeba castellanii (genotype T4), Vermamoeba vermiformis and Naegleria strains including: N. australiensis, N. pagei and N. gruberi in the mineral springs of Guilan Province, northern Iran. In the present study pathogenic N. fowleri was not detected and there were no significant differences shown between pH value (and temperature) or the presence/absence of Acanthamoeba, Naegleria and Vermamoeba (Table 2). Some studies conducted in these areas have identified waterborne pathogens such as Acanthamoeba castellanii and Vermamoeba verformis (Mahmoudi et al. 2015b; Dodangeh et al. 2018).

Recently, infections due to Acanthamoeba have increased in the world, which is more likely related to the presence of Acanthamoeba in the natural environment, especially in water sources with a variety of prevalence detected (Liang et al. 2010; Saburi et al. 2017). We isolated one type of Acanthamoeba (T4), unlike Edagawa et al. (2009) who found T3, and Huang & Hsu (2010) who found that T15 predominated in the waters surveyed. Acanthamoeba T4 genotype is associated with AK (Acanthamoeba keratitis). Acanthamoeba keratitis is seen among people who use non-sterile waters and salt pills for disinfecting or storing their CLs (Feiz-Haddad et al. 2019b). Among the 47 known species of Naegleria, only N. fowleri has been reported to be pathogenic for human (De Jonckheere 2014). Naegleria fowleri was not detected in the present study; this may be owing to differences in the incubation temperature of the culture plates and/or the number of springs. Moreover, in contrast to the current study, others have demonstrated the presence of N. fowleri in the cooling waters of a Belgian power plant and hot springs in Taiwan (Behets et al. 2007; Tung et al. 2013). To date, one clinical case of N. fowleri has been reported in the country (Movahedi et al. 2012). We isolated two cases of N. australiensis in the present study (Table 3). Naegleria australiensis could be pathogenic to animal models (John & De Jonckheere 1985). Also, we isolated three cases of Vermamoeba vermiformis in the present study (Table 3). Keratitis owing to Vermamoeba and also a case of mixed infection of Vermamoeba vermiformis and Acanthamoeba were reported during previous studies (Lorenzo-Morales et al. 2007; Abedkhojasteh et al. 2015; Hajialilo et al. 2015). Vermamoeba amoebae are also considered as a reservoir for pathogenic microorganisms such as Legionella pneumophila and Pseudomonas (Centeno et al. 1996). Mineral springs, used therapeutically and as tourist attractions, is growing in popularity; which will increase the chance of exposure to these amoebae. To prevent infection and diseases related to free-living amoebae, mineral springs should be periodically checked, in particular during the summer season when these surface waters are used by thousands of tourists. Furthermore, posting of warning signs at recreational mineral springs could be another option for preventing FLA infections.

The results of our study approve the presence of potentially pathogenic Acanthamoeba castellanii (genotype T4), Vermamoeba vermiformis and Naegleria strains including: N. australiensis, N. pagei and N. gruberi in the mineral springs of Guilan Province. Mineral springs may enhance exposure of the amoebae to individuals. Hence, more attention to mineral springs is needed to prevent free-living amoebae mediated diseases.

The authors thank the esteemed group of Parasitology Ahvaz Jundishapur University of Medical Sciences and Guilan University of Medical Sciences. The present study was financially supported by Student Research Committee, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran (Grant No. 98 S 73).

The authors declare that there is no conflict of interests.