Free-living amoebas (FLAs) can cause neurological and ocular complications in humans. Water supplies play a critical role in transmitting FLAs to humans. The aim of the present study was to investigate the presence of FLAs in various aquatic sources including drinking water, stagnant water, and surface water in Alborz province, northern Iran, using morphological and molecular techniques. A total of 70 water samples were collected from 34 drinking waters, 23 surface waters, and 13 stagnant waters. Filtration and cultivation were employed to isolate FLAs. PCR assay was applied by using the genus-specific primers on positive samples. Pathogenicity tests (osmo- and thermo-tolerance properties) were performed for Acanthamoeba spp., positive sample. Considering the morphological criteria, four positive samples of Acanthamoeba sp., three Vermamoeba sp., two mixed Vermamoeba sp. with Vahlkamfiids, and one mixed Acanthamoeba sp. with Vahlkamfiids were isolated. Five Acanthamoeba sp. isolates were amplified using the JDP primer pairs. Among them, two genotypes, T4 (three isolates) and T5 (two isolates) corresponding to A. lenticulata, were identified. Four V. vermiformis samples were confirmed using the sequencing. This study highlighted the occurrence of potentially pathogenic waterborne FLAs in water habitats associated with high human activity. The results of such research on the prevalence of FLAs, as a human hazard, should be communicated to health policymakers.
Water samples were collected from drinking, surface, and stagnant waters.
According to the morphological assessment, Acanthamoeba, Vermamoeba, and Vahlkamfiids were detected among samples.
The genotypes T4 and T5 (corresponding to A. lenticulata) were characterized.
Free-living amoebas (FLAs) are ubiquitous parasitic protozoa commonly found in different environmental sources such as water, soil, dust, and air (Landell et al. 2013; Plutzer & Karanis 2016; Niyyati & Latifi 2017). FLAs have an amphizoic life cycle and two distinct forms: trophozoite (vegetative form) and cyst (resistant form) (Padzik et al. 2018). Under favourable environmental conditions, trophozoites play an active role in feeding and replicating (Khan 2003), while cysts resist harsh environmental conditions such as ultraviolet (UV) radiation, heat, dryness, the presence of antimicrobial agents, and disinfectant solutions (Coulon et al. 2010).
Acanthamoeba, Naegleria, Balamuthia, and Vermamoeba are the major genera of the medically important FLAs (Muchesa et al. 2016; Fabros et al. 2021). These FLAs cause a variety of neurological and ocular complications by attacking the central nervous system (CNS) and cornea (Lorenzo-Morales et al. 2011; Hajialilo et al. 2015). Acanthamoeba sp. is the most abundant FLA, which is classified into 22 genotypes (T1–T22) based on the small subunit of 18S ribosomal RNA (rRNA) gene (Chelkha et al. 2020; Corsaro 2020). However, T3, T4, and T5 genotypes have been detected from various clinical cases of Acanthamoeba keratitis (AK) and Acanthamoeba encephalitis (Khan 2009; Lackner et al. 2010; Mirjalali et al. 2013). Among the Vahlkampfiidae family, N. fowleri, a thermophilic FLA, is the only human-pathogenic species that can cause the lethal disease, primary amoebic encephalitis (PAM) (Grace et al. 2015). B. mandrillaris is considered to cause a rare (but deadly) neurological disorder known as granulomatous amoebic encephalitis (GAE) (Mungroo et al. 2020). Another FLA, V. vermiformis (formerly called Hartmannella vermiformis), is less commonly isolated in clinical samples compared to environmental specimens (Delafont et al. 2018; Scheid 2019; Siddiqui et al. 2021).
The distribution of these potentially pathogenic FLAs in various water sources such as drinking water and surface water poses a health hazard to humans. The Karaj river provides drinking water for Alborz province and some regions of Tehran (capital of Iran). In addition, besides the agricultural development in Alborz, this river is a popular weekend summer resort. All water sources that were investigated in the present study have different uses including agriculture, industry, residential, and recreational activities. Therefore, the purpose of the present study was to investigate the occurrence of FLAs isolated from various aquatic sources including drinking water, stagnant water, and surface water in Alborz province, northern Iran, using morphological and molecular techniques.
MATERIALS AND METHODS
Sampling, culture, microscopy detection
This cross-sectional study was carried out between September and November 2020. A total of 70 water samples were collected from 34 drinking waters (eight hospitals, seven pharmacies, seven sports clubs, seven shopping centres, three optometry clinics, and two houses), 23 surface waters (public park waters, river waters, and canal waters), and 13 stagnant waters (open and closed reservoir waters) sources at cities in Alborz province, northern Iran (Figure 1). All drinking water samples were collected from tap waters. Surface waters were from artificial streams, which pumped up to parks' pools (in the case of public park waters) and natural waters (in the case of river and canal waters). The north belt of Karaj is limited by mountains and there are rivers and canal waters, which bring waters that originate from these mountains. In some parts, these rivers and canals are near to cities, and we collected samples from these parts. Sampled surface waters are not affected by wastewater, at least at the sampling sites or upstream areas. Stagnant waters were collected from artificially open and closed reservoir waters. The stagnant waters are untreated and are from surface, well, and raining waters that are collected in a container for further purposes such as irrigation.
After collecting samples, 1,000 mL of each water sample was filtered using nitrocellulose filter papers (Millipore sterile, 0.22 mm pore size and 45 mm diameter), cultivated onto 1.2% non-nutrient agar (NNA) covered by heat-killed Escherichia coli, as previously described (Niyyati & Rezaeian 2015). Cultivated plates were then incubated at 37 °C and monitored daily for 30 days under a light microscope. Positive samples were sub-cultured onto fresh NNA plates in order to achieve a pure culture of the targeted amoebae. Considering Page keys (Page 1988), isolated FLAs were specified to the genus level using morphological criteria.
At first, 103 trophozoites/plate were coated with mannitol-free E. coli and transferred onto the centre of the NNA plate as a control. Then, 103 of each positive isolate were transported to the centre of the plates by coating the NNA plate with 0.5 and 1 M mannitol concentrations and E. coli suspension. Subsequently, the plates were incubated at 30 °C for up to 10 days, and the growth of FLAs at 24, 48, and 72 h was appraised. Trophozoites or cysts were counted from five microscopic areas of approximately 20 mm from the centre of each plate at ×10 microscopic magnification. Finally, the presence of proliferation was evaluated as (+) positive, and the absence of growth (−) as negative (Aykur & Dagci 2021).
For this purpose, 103 trophozoites of FLAs were transferred to the centre of E. coli coated NNA plates. Then, these plates were incubated at 30 °C as a control, 37, and 42 °C for up to 10 days, and were evaluated after 24, 48, and 72 h of the incubation. Subsequently, proliferation was evaluated as described in the osmo-tolerance assay (Aykur & Dagci 2021).
DNA extraction, PCR amplification, and sequencing
Cultivated FLAs were harvested, washed by sterile phosphate-buffered saline (PBS, pH=7.4), and were precipitated by centrifuging for 5 min at 2,000 rpm. Afterwards, DNA extraction was performed by the total DNA extraction kit (Yekta Tajhiz Azma, Iran), according to manufacturer's instructions (YKA, Iran). Four sets of primers were applied in order to detect various FLAs including Acanthamoeba spp. (JDP1,2 primer pairs: F5′-GGCCCAGATCGTTTACCGTGAA-3′ and R5′-TCTCACAAGCTGCTAGGGAGTCA-3′) (Schroeder et al. 2001), B. mandrillaris (Balspec16S primer pairs: F5′-CGCATGTATGAAGAAGACCA-3′ and R5′-TTACCTATATAATTGTCGATACCA-3′) (Booton et al. 2003), Vahlkampfiids (ITS1,2 primer pairs: F5′-GAACCTGCGTAGGGATCATTT-3′ and R5′-TTTCTTTTCCTCCCCTTATTA-3′) (Pélandakis & Pernin 2002), and V. vermiformis (NA1, 2 primer pairs: F5′-GCT CCA ATA GCG TAT ATT AA-3′ and R5′-AGA AAG AGC TATCAATCT GT-3′) (Lasjerdi et al. 2011). PCR reactions were performed in a total volume of 30 μL containing 15 μL of 2X red master mix (Ampliqon, Denmark), 10 ρM of each primer, DNA (10 ng), and distilled water. The thermal cycling profile was set as a pre-denaturation step at 94 °C for 3 min, followed by 35 repetitions at 94 °C for 35 s, and annealing steps were at 56, 56, 56, and 58 °C for 1 min (for Acanthamoeba spp., Vahlkampfiids, Balamuthia spp., and Vermamoeba spp., respectively), and 72 °C for 1 min.
All amplicons were electrophoresed by 1.5% agarose gel, stained with a solution of ethidium bromide, and visualized under a UV transilluminator. The sequencing was conducted using an automated sequencer. To specify genus, species, and genotypes, obtained sequences were compared to the Basic Local Alignment Search Tool (BLAST) against available sequences in the GenBank database.
Evolutionary analyses were conducted in MEGA X. The evolutionary analysis was inferred by using the maximum-likelihood (ML) method and the Kimura two-parameter model. Bootstrap with 1,000 replications was employed to test the reliabilities of the tree. Reference sequences retrieved from the GenBank database were also included in the phylogenetic tree.
Morphological characteristics of the detected amoebas
FLAs were detected in 10 (14.30%) out of 70 water samples including drinking waters (seven isolates), surface waters (two isolates), and stagnant waters (one isolate) (Table 1). Considering the FLA morphological characteristics using Page keys (Page 1988), four positive samples of Acanthamoeba spp., three Vermamoeba spp., two mixed Vermamoeba spp., with Vahlkamfiids, and one mixed Acanthamoeba spp., with Vahlkamfiids, were grown on plates. All positive samples are summarized in Table 2, according to the sampling location, pH, chlorine concentration, water temperature, and sea level.
Molecular detection and sequencing
All cultured positive samples were surveyed by PCR assay. Among them, five Acanthamoeba spp. and four Vermamoeba spp. were amplified and showed band sizes of 450 and 700 bp, respectively (Table 3). As shown in Table 3, from three mixed-FLA plates, only two plates were PCR positive. In addition, each of these two plates was positive for one FLA (one plate for Acanthamoeba and another for Vermamoeba). Despite several attempts, a mixed sample containing Vermamoeba spp. and Vahlkamfiids was not successfully sequenced due to high bacterial and fungal contaminations. It is important to mention that after conducting a molecular assay, B. mandrillaris and Naegleria spp. were not detected in samples.
The pathogenicity tests were assessed by the cultivation of isolates in different temperatures (37 and 42 °C) and osmolarity ranges (0.5 and 1 M mannitol). The growth of isolates at high temperature (at 42 °C) and high osmolarity (1 M mannitol) was considered as pathogenic potency. Growth at 37 °C and 0.5 M osmolarity were considered as low pathogenicity. The thermo-tolerance and osmo-tolerance properties of the positive samples are listed in Table 3.
The sequencing results for Acanthamoeba sp. revealed the presence of the genotypes T4 (n=3, accession numbers MZ955619, MZ955620, and MZ955621) and T5 corresponding to A. lenticulata (n=2, accession numbers MZ955617 and MZ955618) (Table 3). All four V. vermiformis samples were also confirmed (accession numbers MZ955622, MZ955623, MZ955624, and MZ955625) (Table 3).
This study reports the occurrence of waterborne FLAs belonging to the Acanthamoeba sp., T4 and T5 genotypes, and V. vermiformis in water sources of Alborz province. In Iran, the previous epidemiological studies have shown that Acanthamoeba sp. has a higher prevalence than other FLAs in both clinical and environmental samples (Hajialilo et al. 2015; Mahmoudi et al. 2015, 2021; Saburi et al. 2017). One of the most important reasons for the high occurrence of Acanthamoeba sp. is its high capability to adapt to harsh environmental conditions (Aksozek et al. 2002). Previous studies have suggested that using that tap water for washing contact lenses is a major risk factor for AK development (Lorenzo-Morales et al. 2005; Shoff et al. 2008; Koltas et al. 2015). The most reported cases of AK are related to people who wear soft contact lenses, which occur due to inappropriate maintenance of their lenses (Lindsay et al. 2007). According to the results of this study, seven out of 10 samples, which were contaminated with FLAs, belonged to drinking water. In this regard, cleaning contact lenses with distilled water or non-sterile water is considered a risk factor for AK (Khan 2006; Niyyati & Rezaeian 2015). Based on the published literature, two isolated genotypes in this study (T4 and T5) are considered as the genotypes, which were reported in patients with AK (Ledee et al. 2009; Niyyati et al. 2010; Omaña-Molina et al. 2016). The presence of A. lenticulata T5 genotype was associated with acute granulomatous encephalitis in an immunocompetent patient (Lackner et al. 2010). Moreover, a fatal case of disseminated acanthamoebiasis caused by A. lenticulata (genotype T5) has been reported in a 39-year-old heart transplant recipient (Barete et al. 2007). There are reports of the T5 genotype from mucosal tissue of immunocompromised individuals, which may predispose the presence of this genotype in ocular and cerebral involvement (Memari et al. 2015, 2017; Niyyati et al. 2017; Tananuvat et al. 2019). Thereby, immunocompromised patients and people wearing contact lenses need to be fully aware of the transmission and pathogenesis to prevent infections due to Acanthamoeba sp.
As shown in this study, one of the most commonly identified FLAs was V. vermiformis. However, the pathogenicity of V. vermiformis has not yet been fully determined, but some case reports of this FLA have been reported in patients with keratitis (Lorenzo-Morales et al. 2007; Abedkhojasteh et al. 2013; Scheid et al. 2019) and painful ulcers close to the eye (Scheid et al. 2019). Lorenzo-Morales et al. (2007) reported a mixed infection of Acanthamoeba sp. and Hartmannella from a patient with keratitis. However, more in-depth studies should be performed to gain a better insight into the status and pathogenicity of this FLA.
B. mandrillaris was not isolated in the collected samples of this study. In accordance with our study, B. mandrillaris has not been reported previously in water samples from Semnan, Iran (Javanmard et al. 2017). However, in previous studies from Iran, the occurrence of B. mandrillaris has been reported in hospital dust, soils of recreational areas, and hot springs (Niyyati et al. 2009; Latifi et al. 2016). Therefore, further investigations are needed to assess the exact niches of B. mandrillaris in the country.
In the present study, none of the 47 species of Naegleria spp. were reported using the ITS-based sequencing technique. One of the most important reasons for non-isolation of Naegleria spp. in our study can be the incubation temperature of the culture, as most Naegleria species are thermophilic and grow in temperatures of 38–40 °C (Jahangeer et al. 2020; Saberi et al. 2020). Therefore, incubation of plates at different temperatures should be tried to detect Naegleria spp. in future studies.
Importantly, the presence of FLAs in water samples, which are routinely used by humans, increases the risk of contamination. In the current study, drinking waters collected from hospitals, drug stores, eye clinics, sports clubs, and shopping centres were contaminated with FLAs. Notably, all drinking waters in these places were collected from tap waters that are used not only for drinking but also for washing hands, dishes, hospital sheets, etc. On the other hand, three isolates JA3, JA4, and JA5, which were obtained from drinking waters from optometry clinics, hospitals, and sports clubs, respectively, were osmo and thermotolerant. This feature of isolated FLAs signifies the pathogenic potential of isolated strains, which increases the medical and public health importance of FLAs in these places.
Phylogenetic tree represented similarity of the isolates JA4 and JA5 (which were isolated from hospitals and sports clubs, respectively) with Acanthamoeba sp., T4 genotypes, which were reported from contact lens, stagnant waters, and AK (Hajialilo et al. 2015; Golestani et al. 2018; Hussain et al. 2020). This genetic similarity suggests the potency of isolates to cause acanthamoebiasis in hospitalized patients or those people who use contact lenses.
In conclusion, this study highlighted the occurrence of potentially pathogenic waterborne FLAs in different water supplies, especially drinking water, in places with high human activity such as sports clubs, hospitals, and eye clinics. Further studies to evaluate the niches of B. mandrillaris and N. fowleri are important in Iran. Results of such research on the distribution, relevance, and clinical significance of FLAs should be communicated to health policymakers.
The authors thank all members of the Department of Parasitology and Mycology and Foodborne and Waterborne Diseases Research Center for their support.
M.N., H.M., and E.J. conceived and designed the experiments. E.J. and M.F. performed the experiments. M.N., H.M., and A.T. analysed the data. H.M. contributed reagents/materials/analysis/tools/positive samples. A.T., M.N., H.M., and P.K. wrote the paper. All authors read and approved the final version of the manuscript.
ETHICS APPROVAL AND CONSENT TO PARTICIPATE
All procedures performed in this study were in accordance with the ethical standards (IR.SBMU.MSP.REC.1399.26) released by the Ethical Review Committee of the Shahid Beheshti University of Medical Sciences, Tehran, Iran.
CONSENT FOR PUBLICATION
All authors declare that they have seen and approved the submitted version of this manuscript.
AVAILABILITY OF DATA AND MATERIAL
All generated data from the current study are included in the article.
This study was financially supported by the Shahid Beheshti University of Medical Sciences under grant no. 20742.
CONFLICT OF INTEREST
The authors declare that they have no conflict of interest.
DATA AVAILABILITY STATEMENT
All relevant data are included in the paper or its Supplementary Information.