Surface water contamination with Acanthamoeba spp. in Ilam city, Iran

Acanthamoeba , a widely distributed free-living amoeba with 20 genotypes identi ﬁ ed through rRNA gene sequencing, exhibits varying degrees of pathogenicity in ﬂ uenced by its genotype. This study focuses on assessing the prevalence of Acanthamoeba species in the surface waters of Ilam, located in western Iran, utilizing morphological analysis and sequencing of the 18S rRNA gene through the PCR method. A total of 50 water samples were collected from various regions within Ilam city, situated in the southwest of Iran. To isolate Acanthamoeba parasites from the samples, a culture method was used, and all utilized culture media were scrutinized through microscopic and molecular techniques. The parasite ’ s genotype was determined by sequencing a 500-bp fragment of the 18S rRNA gene. Using microscopic and molecular methods, 19 and 16 water samples tested positive, respectively. The 18S rRNA sequences revealed that the isolates belonged to the T4, T2, and T11 genotypes. This study emphasizes the presence and inclination for close contact with highly pathogenic genotypes of Acanthamoeba in the surface waters of Ilam City.


INTRODUCTION
Free-living amoebae are opportunistic protozoans distributed globally in a wide range of environmental sources including water, soil, air, hot springs, and urban water supply systems (Siddiqui & Khan 2012;Padzik et al. 2019).Acanthamoeba belongs to the Acanthamoebidae family, exhibiting both trophozoite and cystic forms throughout its life cycle (Visvesvara 1991;Panjwani 2010;Eslamirad et al. 2020).Characterized by their high resistance to various environmental factors, they maintain their pathogenicity even after prolonged exposure to temperature changes, pH variations, and chlorination (Mosayebi et al. 2014;Behniafar et al. 2015).Granulomatous encephalitis, Acanthamoeba keratitis, and cutaneous Acanthamoeba represent the most significant clinical manifestations resulting from infestation by the pathogenic genotypes of the Acanthamoeba parasite in humans (Nazar et al. 2011).In addition, complications such as sinus infections, dermatitis, and the development of severe skin ulcers are associated with this protozoan, particularly in individuals with compromised immune systems (Niyyati et al. 2016).Morphological taxonomic studies have identified 24 species of Acanthamoeba.However, the rRNA gene sequence confirms only 20 genotypes (T1-T20) (Hosseinbigi et al. 2012), with the T3 and T4 genotypes exhibiting the highest pathogenicity in their hosts (Booton et al. 2005).Genotyping of clinical isolates of Acanthamoeba has revealed that the T4 genotype is accountable for 90% of amoebic keratitis.The varying pathogenicity observed among different Acanthamoeba genotypes and their roles in disease prognosis, particularly in cases of amoebic keratitis, underscores the significance of Acanthamoeba genotyping in the diagnostic and treatment processes (Khan et al. 2002).In light of the amoeba's close association with human habitation, its ecological characteristics, widespread presence in the environment, and the rising frequency of immune diseases, as well as common recreational activities such as swimming in seasonal water, utilizing surface water in the region for drinking, washing, and bathing, it becomes crucial to consider the multitude of surface water sources.These sources may potentially be contaminated by resilient cysts of the parasite, carried through sand and dust storms originating from neighboring countries (Iraq, Kuwait, and Saudi Arabia).The assessment of the Acanthamoeba fauna in the environment holds significant implications for public health.This study, utilizing morphological analysis and sequencing of the 18S rRNA gene through the PCR method, aims to investigate the prevalence of Acanthamoeba species in the surface waters of Ilam City, located in the western part of Iran.

Study areas and sampling
A total of 50 samples (Table 1) were collected from diverse surface water sources, encompassing surface water, springs, pools, city square ponds, seasonal water, drinking water sources, and sewage, across different regions of Ilam City in the western part of the country (Figure 1).Ilam province, acknowledged as one of the tropical regions of the country, exhibits three distinct climate types due to variations in altitudes, precipitation, and temperature.The mountainous areas to the north and northeast experience cold climates and prolonged winters, while the plains of the west and southwest endure a dry, hot climate.Other regions feature temperatures ranging from temperate to warm, characterized by hot summers and mild winters.Recorded temperatures had ranged from a high of 47.0 °C or 116.6 °F on 20 August to a low of À15.0 °C or 5.0 °F on 5 February, with an annual precipitation range between 200 mm (7.9 in) and 450 mm (18 in).The samples, collected randomly using 500 mL sterile bottles, were subsequently transferred to the laboratory.All research procedures were conducted at the Department of Parasitology and Mycology of Ilam University of Medical Sciences.The sample size was determined through ratio analysis, taking into account the standard deviation from previous studies, and the results were analyzed using SPSS 19.

Samples filtration
All containers were shaken for 3 min to ensure homogeneity of the contents, and water filtration was performed using a nitrocellulose filter with a pore diameter of 45 μm, coupled with a vacuum pump.

Acanthamoeba parasite culturing
After filtration, the nitrocellulose filters were placed upside down on the surface of a sterile non-nutrient agar medium coated with Escherichia coli bacteria.To mitigate culture media evaporation, the plates were sealed with parafilm and incubated at 25 °C for 24 h.Subsequently, the filters were removed from the culture medium to prevent the proliferation of fungal saprophytes.Microscopic examination for the presence of Acanthamoeba trophozoites, particularly along the culture medium margins, was conducted 4 days after parasite culture initiation.Given the slow growth of certain Acanthamoeba species, the cultured media were incubated for one month, with microscopic assessments performed at various intervals.

Amoeba cloning
To eliminate potential contamination by bacteria, fungi, and free worm larvae, amoeba cloning was executed.Under a light microscope, Acanthamoeba cysts devoid of bacterial or fungal colonies in their vicinity were carefully excised using a sterile scalpel and subsequently transferred to a fresh culture medium.Daily observations were made on the new plates to monitor amoeba growth and detect the emergence of contaminants.Through several successive passages in culture media, amoeba plates obtained were ultimately free from any contamination.
Isolation of Acanthamoeba trophozoites from the culture medium The isolation of Acanthamoeba from the culture medium involved the following steps: 5 mL of phosphate-buffered saline (PBS) buffer was added to the plates, and the medium was sectioned using a sterile scalpel blade.The plate surface was washed 10 times with a sterile Pasteur pipette until cysts and trophozoites floated in the PBS.The resulting mixture was transferred to 1.5 mL microtubes and centrifuged at 200g for 10 min.Subsequently, the supernatant was discarded, and the sediments were washed three times with PBS to eliminate any agar residue.The isolated parasites were then collected and stored at À20 °C for morphological and molecular identification.

Morphological diagnosis of Acanthamoeba
A volume of Giemsa stain and 40 volumes of the isolated sample were mixed and stained for 12 min.Following this, wet smear slides were prepared and examined under a light microscope Â10 and Â40 lenses.

Molecular detection
Genomic DNA was extracted using the Favor Prep DNA Isolation Mini Kit according to the manufacturer's instruction.Primers (18S rRNA) were designed by Schroeder and produced a 500-bp fragment (Table 2).PCR was performed in a final reaction volume of 25 μl using 12.5 μl of Taq Master Mix (Yekta Tajhiz Azma Company, Iran; which contained 0.2 mM dNTPs, 0.1units/μl Ampliqon Taq DNA polymerase), 1.5 μl of each primer (10 pmol), and 4 μl of DNA template, bringing the final volume with the sterile distilled water (5.5 μl) to 25 μl.The Bio RAD (New York, USA) thermocycler was used for the PCR reactions.The temperature profile was an initial denaturation at 94 °C for 5 min, followed by 40 cycles of denaturation at 94 °C for 60 s, annealing at 57 °C for 60 s, extension at 72 °C for 60 s, and a final extension at 72 °C for 5 min.The PCR product was electrophoresed on 1.5% agarose gel stained with DNA-safe stain dye (Pishgam Biotech Co., Tehran, Iran) for 45 min at 100 V and visualized using a UV transilluminator.

Sequencing, phylogenic and data analysis
The PCR products were sequenced in one direction using a forward primer (Takapozist Co., Tehran, Iran).Subsequently, the generated sequences were manually organized and compared to analogous Acanthamoeba sequences in the Gene Bank utilizing the Chromas software version 2.5.0.Alignments were performed with similar sequences derived from Acanthamoeba species isolated from various regions.Distances between the sequences were calculated, and a phylogenetic tree was constructed using MEGA version 7 software, and the maximum likelihood method with a generation of 1,000 bootstrapped replicates.

Diagnosis of Acanthamoeba using light microscopy
All culture plates were meticulously examined daily under a light microscope.Clear spots detected on the culture medium were interpreted as the presence of trophozoites.Employing the Giemsa stain method, Acanthamoeba endocysts appeared stained blue, while ectocysts exhibited a very pale to colorless appearance.On the fifth day after culture initiation, 17 out of the 50 plates (34%) tested positive.All cysts displayed a distinct wall with spherical, polyhedral, and star-shaped endocysts (Figure 2).Due to the slow growth rate of certain Acanthamoeba species, two plates required an additional 3 weeks to yield positive results.A total of 19 samples (38%) tested positive within a 4-week culture period on non-nutrient agar media (Table 1).

PCR analysis and sequencing
The primers amplified 500 bp fragments during the PCR amplification.Sixteen (32%) samples yielded the expected 500 bp amplicons through the molecular method (Figure 3).Upon comparing the obtained sequences in this study with those registered in the World Gene Bank, it was determined that the isolates belonged to the T2, T4, and T11 genotypes, with frequencies of 12.5, 75, and 12.5%, respectively (Table 1).

BLAST and phylogenetic analysis
In BLAST analysis, the T2 genotype sequences generated here exhibited 99-100% identity with sequences isolated from Tehran clinical samples (EU934072), Kerman (KC694193), and the United States (AF019063).Moreover, the T4 genotype sequences of this study showed 99-100% identity with sequences isolated from Varamin, Iran (MF806034.1),Chile (KT892904), and Poland (KR259814).The T11 genotype sequences exhibited 98% identity with sequences isolated from Italy (KJ094683), the United States (AF333608), and Thailand (KX069000).The results of the phylogenetic analysis confirmed the relationship between the isolates of this study with the other studies and species registered in the gene bank.Based on the phylogenetic analysis of the 18S rRNA gene sequence, the isolated samples of the present study were divided into three separate branches.In the first branch, AC3, AC4, AC6, AC8, AC9, AC10, AC12, and AC13 isolates with T4 genotypes registered in GenBank were placed next to each other.AC1 and AC7 with T11 genotypes separated into a new branch, and then AC2 and AC5 constructed a branch with Acanthamoeba Palestinians' T2 genotypes (Figure 4).

DISCUSSION
The findings of this investigation provide evidence for the presence of Acanthamoeba in surface water within Ilam, western Iran.These results align with prior studies that have successfully isolated this amoeba from various regions across Iran (Nazar et al. 2011).Additional studies have reported higher frequencies of Acanthamoeba contamination in the hot springs of Ilam and stagnant surface waters of Qazvin compared to the outcomes observed in our study (Hosseinbigi et al. 2012;Niyyati et al. 2016).The variation in Acanthamoeba prevalence among the studied areas can likely be attributed to distinctions in the sources of water samples and the environmental conditions specific to each region.When comparing molecular and microscopic examination methods, molecular techniques demonstrated greater sensitivity and specificity in identifying Acanthamoeba genotypes (Khan et al. 2002;Booton et al. 2005), and the current study reinforces these data, as alignment of the 18S rRNA gene sequences obtained herein with those registered in the GenBank reveals that the T4 genotype of Acanthamoeba predominates in the water resources of Ilam.Consistent with previous research, it is well established that the T4 genotype serves as the most prevalent causative agent of both amoebic keratitis and granulomatous Acanthamoeba encephalitis (Zhang et al. 2004;Sheng et al. 2009;Liang et al. 2010), and this matter might be related to the higher prevalence (Shoff et al. 2008), virulence, and potential transmissibility associated with the T4 genotype in the environment (Khan 2006).The T4 genotype has a higher tendency to bind to the host cells compared to the T2, T3, and T11 genotypes (Alsam et al. 2003).The urban pools yielded the most pathogenic genotypes in this study, specifically T4 and T2.This underscores the significance of determining genotypes in both clinical and environmental samples.In the context of Iran, T4 emerges as the dominant genotype, alongside reports of T2, T3, T5, T6, T9, T11, T13, and T15 genotypes (Badirzadeh et al. 2011;Spotin et al. 2017).Previous investigations have highlighted a close relationship between the T11 and T4 genotypes and supported and validated this established association (Walochnik et al. 2000).The prevalence of the free-living amoebae in the treated water network is observed to be lower compared to untreated surface water.The chlorination system is effective in destroying amoebic trophozoites.However, it is noteworthy that certain studies have reported instances of contamination in treated and chlorinated water with this protozoan (Latifi et al. 2015).Hence, the proximity of surface water to potential pollution sources likely constitutes a major factor contributing to the higher contamination levels with the Acanthamoeba parasite, distinguishing it from other water sources.Considering that the Acanthamoeba parasite is highly pathogenic in all climatic regions and can cause many pathogenic lesions including eye, skin, lung, mucosal lesions, etc., in all people of the society, the results of this study can be a serious warning for health officials and clinicians to design a plan to prevent and treat this disease and be used as a reliable document by healthcare decision-makers.

CONCLUSION
In conclusion, this study revealed a widespread prevalence of Acanthamoeba in various water resources.The analysis of the 18S rRNA gene sequence further confirmed the presence of the highly pathogenic T4 genotype, demonstrating its highest frequency in the surface water of Ilam.These findings underscore the importance of continued monitoring and awareness of Acanthamoeba contamination in water sources for effective public health management.

Figure 2 |
Figure2| To isolate the Acanthamoeba from samples, the nitrocellulose filters, after filtration, were placed upside down on a sterile nonnutrient agar medium, coated with Escherichia coli bacteria.The cysts were stained with the Giemsa stains and observed under a light microscope.The endocysts of Acanthamoeba cysts were stained blue in spherical, polyhedral, and star shapes, and the ectocysts were very pale to colorless.

Figure 4 |
Figure 4 | The phylogenetic tree of Acanthamoeba sp.Based on the 18S rRNA gene constructed by the maximum likelihood method based on the Tamura-Nei model using MEGA 7 software.The accession numbers of sequences used for the tree's construction are shown in parentheses, and the sequences generated in this study are marked by an asterisk.

Table 1 |
Data of isolates obtained from regions of Ilam: microscopic and molecular results

Table 2 |
Primers used for amplification of the 18S rRNA fragment