Isolation and genotyping of Acanthamoeba species and Vahlkampfiidae in the harsh environmental conditions in the centre of Iran

Free-living amoebae (FLAs) are opportunistic and pathogenic protozoans that live independently in soil or aquatic habitats (Saburi et al. 2017; Fabros et al. 2021). They belong to the genera Acanthamoeba, Balamuthia, Naegleria, Vermamoeba, and Sappinia (Trabelsi et al. 2012). These amoebae are potentially pathogenic to humans and cause symptoms of meningoencephalitis, encephalitis, and acanthamoeba keratitis (AK) (Lorenzo-Morales et al. 2015; Król-Turmińska & Olender 2017). In recent years, most studies have focused on identifying and determining the genotype of Acanthamoeba species, because previous studies have shown that the incidence rate of AK has increased in Iran and these cases continue to rise (Haddad et al. 2019; Hajialilo et al. 2019). In addition to AK, the medical importance of Acanthamoeba species is related to the infection of other organs of the brain, urinary tract, and skin (Kot et al. 2021; Saberi et al. 2021; Park et al. 2023). It should be noted that only one primary amoebic meningoencephalitis with the causative agent Naegleria fowleri has been reported in Iran (Movahedi et al. 2012).

Acanthamoeba species were initially divided into three morphological groups (I, II, and III) based on the size and structure of ecto- and endocyst (Pussard 1977). The genus Acanthamoeba was classified with 23 genotypes (T1–T23) based on the analysis of the small subunit of the ribosomal RNA gene (SSU-18S-rRNA) (Corsaro 2022). According to shreds of evidence, genotypes of Acanthamoeba species in Iran include T2, T3, T4, T5, T11, T13, and T15 (Spotin et al. 2017). The T4 genotype is the most common genotype identified in clinical and environmental samples (Maciver et al. 2013).

Qom Province, located in the central part of Iran, has several tourist attractions due to the second destination of religious tourism, palaces, historical villages, mosques, gardens, and the famous Salt Lake Desert. The Qom River or Qom Roud is a large river (approximately 400 km) that receives its water from the Zagros Mountains and mounds into the Namak Lake in Qom Province. In the summer season, people go around the Qom Roud for fun and an exciting time. One of the most popular activities during spring and summer is swimming, which brings great joy to children. As regards the amoeba being resistant to harsh conditions, the present research was conducted to identify and determine the genotype of FLAs in the Qom Roud in Qom Province.

The School of Public Health's laboratory investigated the physicochemical features of water samples, and they provided us with the values. A pH meter was used to measure pH, a conductivity meter device was used to measure EC and TDS, and a Flame photometer device was used to measure Na⁺ and K⁺. It should be noted that the titration method was used to measure TH, HCa⁺, HMg, Cl⁻, and ALK. Finally, ⁠, ⁠, and parameters were measured using a DR 5000™ UV–Vis Spectrophotometer.

According to Page (1988), isolated amoebae were identified at the genus level. Acanthamoeba cysts were identified by the presence of a double wall and consisted of ectocyst and endocyst, or trophozoites by the presence of acanthopodia. Vahlkampfiidae cysts are spherical, containing a single nucleus and a double wall with pores and wormy-shaped trophozoites.

All positive morphological plates were subjected to the molecular method. DNA was extracted using a DNA extraction kit (Yekta Tajhiz Azma, Tehran, Iran) according to the manufacturer's instructions. Two sets of specific primers in order to amplify Acanthamoeba species (Forward: 5′-GGCCCAGATCGTTTACCGTGAA-3′ and Reverse: 5′-TCTCACAAGCTGCTAGGGAGTCA-3′) (Schroeder et al. 2001) and Vahlkampfiidae (Forward: 5′-GAACCTGCGTAGGGATCATTT-3′ and Reverse: 5′-TTTCTTTTCCTCCCCTTATTA-3′) were used as previously described (Pélandakis & Pernin 2002). Amplification reactions were set for a total volume of 25 μl, containing 12.5 μl master mix (Ampliqon Co., Denmark), 1 μl forward and reverse primers (10 pmol), 3 μl DNA templates (70 ng), and 8.5 μl nuclease-free water. The temperature program included a first cycle of denaturation at 94 °C for 5 min, 35 cycles of denaturation at 94 °C for 45 s, annealing steps at 56 °C for 1 min (for both Acanthamoeba and Vahlkampfiidae), and extension at 72 °C for 45 s, then a final extension step at 72 °C for 5 min. The PCR products were confirmed by imaging on 1.5% agarose gel (Invitrogen, Life Technologies GmbH, Leipzig, Germany) stained with SYBR™ Safe DNA Gel Stain (Thermo Fisher Scientific, Waltham, MA, USA) (Javanmard et al. 2017). Positive and negative controls always monitored the reactions.

PCR products of the Acanthamoeba and Vahlkampfiidae isolates were purified and sequenced using the Sanger sequencing method in both directions, by Pishgam Biotech Company, Iran. Different software used for genetic data analysis are as follows. Chromas version 2.6, for viewing and editing chromatograms from sequences, BioEdit, version 7.0.5 used for multiple alignments, and BLAST (http://blast.ncbi.nlm.nih.gov) for finding sequence similarity with GenBank sequences, and MEGA X for drawing the phylogenetic tree. To estimate phylogenetic trees, we selected the Neighbor-Joining method using Kimura 2-parameter models in MEGA X. In addition, bootstrap resampling analysis (1,000 replications) was used to assess branch confidence in clades in each tree (Kumar et al. 2018). Furthermore, Balamuthia mandrillaris was used as an outgroup to construct the phylogenetic tree.

To determine the pathogenic potential of the positive isolates, thermal and osmotolerance tests were used following the protocol of Khan et al. (2001). Thermotolerance of Acanthamoeba was measured as survival at two temperatures (37 and 44 °C) and osmotolerance was determined as the capacity of the culture to D-Mannitol (Merck, Darmstadt, Germany) in two plates at different molarities (0.5 and 1 M).

The value of the physicochemical features of water samples in the sampling sites is tabulated in Table 1.

Table 1

The amounts of each recorded physicochemical feature in the studied location during sampling

Parameter .	Unit .	Result .	Optimal maximum .	Maximum allowed .
pH	μS/cm	7.37	–	6.5–8.5
EC	mg/l	1,540	250	400
TDS	mg/l CaCO3	4,616	1,000	1,500
TH	mg/l CaCO3	1,600	200	500
HCa+	mg/l CaCO3	960	300	–
HMg	mg/l	640	30	–
	mg/l	1.1	–	50
	mg/l	1,380	250	400
Na+	mg/l	1,100	200	200
K+	mg/l	14.3	–	12
Cl−	mg/l	1,540	250	400
ALK	mg/l CaCO3	248	–	–
	mg/l	0.8	–	–
Saltiness	mg/l	2,782	–	–

Parameter .	Unit .	Result .	Optimal maximum .	Maximum allowed .
pH	μS/cm	7.37	–	6.5–8.5
EC	mg/l	1,540	250	400
TDS	mg/l CaCO3	4,616	1,000	1,500
TH	mg/l CaCO3	1,600	200	500
HCa+	mg/l CaCO3	960	300	–
HMg	mg/l	640	30	–
	mg/l	1.1	–	50
	mg/l	1,380	250	400
Na+	mg/l	1,100	200	200
K+	mg/l	14.3	–	12
Cl−	mg/l	1,540	250	400
ALK	mg/l CaCO3	248	–	–
	mg/l	0.8	–	–
Saltiness	mg/l	2,782	–	–

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Interestingly, four Acanthamoeba strains (R23, R24, R26, and R28) possessed the ability to grow at both 37 and 44 °C and both osmolarity (0.5 and 1 M), which these isolates were considered as highly pathogenic amoebae. On the other hand, Acanthamoeba species growth at a temperature of 37 °C and 0.5 M osmolarity is classified as a strain with low pathogenicity potential (Table 2).

Since the first discovery of the genus Acanthamoeba over nine decades ago (Castellani 1930), subsequently increasing number of species of Acanthamoeba have been globally reported. The clinical significance of Acanthamoeba species should be considered in particular (Marciano-Cabral & Cabral 2003). AK is a painful and sight-threatening infection that negatively affects a patient's quality of life (Varacalli et al. 2021). Recently, Acanthamoeba species have been detected in the bronchoalveolar lavage fluid from immunocompetent patients with chronic respiratory disorders, and urine samples were collected from patients presenting with recurrent urinary tract infections (UTIs) (Saberi et al. 2021, 2022). The prevalence of Acanthamoeba species in the water represents a sanitary risk for humans. Numerous studies have shown that the Acanthamoeba species has been reported abundantly in different environmental sources in Iran (Karamati et al. 2016; Javanmard et al. 2017; Norouzi et al. 2021). The present study shows the presence of Acanthamoeba species and Vahlkampfiidae in harsh environmental conditions. The Qom Roud is a large river in Iran that receives its water from the Zagros Mountains and mounds into Namak Lake. The name Namak Lake is derived from the high salinity of its water. Therefore, the results of this research indicate the presence of Acanthamoeba species and Vahlkampfiidae in difficult environmental conditions with high salinity.

It seems that Acanthamoeba is the most predominant protozoa present in the environment. We reported that 46.66 and 6.66% of isolates were positive for Acanthamoeba species and Vahlkampfiidae, respectively. This finding is in line with the Latifi et al. study, in which Vahlkampfiidae (45.45%) and Acanthamoeba (40.9%) were isolated from hot springs of Mazandaran province, northern Iran (Latifi et al. 2014). A similar observation was made earlier, where Acanthamoeba species (50%) were detected in geothermal rivers in southwestern Iran (Niyyati et al. 2016). It seems that the relatively high abundance of Acanthamoeba species in harsh environmental conditions, such as hot springs or high salinity, is due to the presence of a resistant cyst form in the life cycle of the parasite (Aksozek et al. 2002).

Genotyping of Acanthamoeba helps to identify the different species of this protozoan (Megha et al. 2023). In the current study, the isolates were identified as T4 genotypes by phylogenetic analysis, an approach proven to be useful for the molecular characterization of Acanthamoeba species. Moreover, the phylogenetic analysis of the current Acanthamoeba T4 genotype and N. philippinensis sequences was similar to those of reference isolates. The sequences in the phylogenetic tree were grouped distinctly into two main clusters, one containing the Acanthamoeba and N. philippinensis sequences, as we expected. Interestingly, the current sequences of the T4 genotype showed several different nucleotides in distinct Acanthamoeba isolates. This aspect indicates the genetic variation of the T4 genotype (Megha et al. 2023). Generally, Acanthamoeba genotyping offers valuable information in studying their taxonomy, drug susceptibility, molecular epidemiology, and clinical studies (Ledee et al. 2009; Fuerst 2014).

In the end, according to thermo- and osmotolerance tests, four Acanthamoeba isolates also demonstrated pathogenic potential. Thermo- and osmotolerance experiments are important in comprehending the environmental and physiological features of Acanthamoeba, as well as their potential to cause infections (Khan et al. 2001). However, it is suggested that more research should be carried out to determine pathogenicity (Mirjalali et al. 2013).

The current findings serve as a document for the presence of Acanthamoeba species and Vahlkampfiidae amoeba in the salty water of Qom Roud. Acanthamoeba isolates belonging to the T4 genotype, which is implicated in the majority of Acanthamoeba infections. This river is related directly to human populations and further investigation is needed for genotype distribution, identification, training, and prevention.

All authors contributed to the study conception and design. A.P.A, L.Z.F., and M.F. carried out the experiment. R.S. wrote the manuscript with support from M.N. All authors read and approved the final manuscript.

All relevant data are included in the paper or its Supplementary Information.

The authors declare there is no conflict.