Prevalence of free-living amoebae in five rivers associated with high human activity in Tehran province, Iran Open Access

Free-living amoebae (FLA) are ubiquitous protozoa that can remain viable in harsh environmental conditions (Plutzer & Karanis 2016). These microorganisms have an amphizoic life cycle with two distinct forms: trophozoite (vegetative form) and cyst (resistant form). These microorganisms are frequently reported from environmental and clinical samples (Visvesvara et al. 2007). Vahlkampfiidae, Acanthamoeba spp., Sappinia diploidea, Balamuthia mandrillaris, and Vermamoeba vermiformis are the major human-infecting FLA that have been reported worldwide (Trabelsi et al. 2012).

FLA are responsible for a broad spectrum of diseases, in which Naegleria fowleri and Acanthamoeba spp. are the most important causative agents (Khan 2007; Trabelsi et al. 2012). Primary amoebic meningoencephalitis (PAM) is a lethal condition caused by N. fowleri. Acanthamoeba spp. cause a variety of neurological and ocular complications by attacking the central nervous system and cornea, respectively. The most common clinical form is Acanthamoeba keratitis (AK), which can occur via various routes, such as swimming while wearing contact lenses (Pinna et al. 2017; Diehl et al. 2021; Niederkorn 2021).

Acanthamoeba spp. is one of the most clinically significant FLAs, which could be transmitted to humans via contaminated water sources, particularly those waters linked with high human activity (Salazar-Ardiles et al. 2022). In addition, Acanthamoeba spp. have been isolated from broad environmental sources, suggesting wide potential sources of infections (Fatemi et al. 2023; Moreno-Mesonero et al. 2023). Acanthamoeba spp. are present in most places in the environment (water, soil, and air), are easily attached to different surfaces, and can remain viable in the environment for a long time (Javanmard et al. 2017; Pazoki et al. 2020; Wopereis et al. 2020; Masangkay et al. 2024).

The isolation and identification of FLA are based on their morphological and molecular criteria. The amplification of a discriminative fragment (DF3 region) is a common practice to determine the genus, species, and genotypes (Risler et al. 2013). Accordingly, based on the sequence analysis of the 18S ribosomal RNA (rRNA) gene, more than 20 genotypes have been identified (Diehl et al. 2021). However, the genotype T4 is the most abundant and widespread, and it has been frequently reported in both clinical and different environmental samples (Diehl et al. 2021).

Numerous studies have investigated the presence and genetic distribution of FLA in environmental samples in Iran (Javanmard et al. 2017; Pazoki et al. 2020; Fatemi et al. 2023). However, little is known about FLA from river water, particularly those that are used for recreational purposes. The current research aimed to investigate FLA in five major rivers of Tehran province, Iran, which are located near numerous restaurants, motels, and natural parks. These rivers also provide water for irrigating the farmlands downstream.

The present study was conducted on 60 water samples collected from five main rivers in Tehran province.

A total of 60 water samples, with 12 samples collected from each river, were collected based on the Environmental Protection Agency (EPA) recommendations (Method 1694). The sampling period was from February 2023 to January 2024, including four seasons: Spring (April, May, and June), Summer (July, August, and September), Autumn (October, November, and December), and Winter (January, February, and March). Briefly, 5 L of samples per month were taken from 30 cm below the surface of each river with a sterile container. Samples were immediately transported to the Foodborne and Waterborne Diseases Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran for parasitological and molecular investigations. Environmental and physical data of the sampling sites and water samples, including water temperature, environmental temperature, solar radiation angle (measured in watts per square meter, W/m²), and the pH of each sample, were recorded in an Excel format during each sampling.

For analysis of FLA, 1 L of samples were filtered using a six-branch filtration system (Sartorius, Goettingen, Germany) and a sterile 47-mm cellulose nitrate membrane (Sartorius, Goettingen, Germany) with a pore size of 0.45 μm. For each run, a negative control was 5 L of sterile water. The filter paper containing sediments was placed side down on 1.2% non-nutrient agar (NNA, Difco, Sparks, MD, USA) together with a lawn of heat-inactivated Escherichia coli strain k12 (Fatemi et al. 2023). For samples with high turbidity and to avoid the obstruction of filter papers, 5 L of samples were transferred to a sterile graduated cylinder for 48 h. This step allows the larger suspended particles to settle at the bottom of the cylinder due to gravity. Afterward, 1 L of supernatants was filtered as described above (Andalib et al. 2022). All plates were incubated at room temperature (26 ± 3 °C) and monitored every 48 h for FLA growth over 1 month. Positive plates were then sub-cultured to achieve a pure culture of the targeted amoebae. Plates were then sealed with parafilm to prevent the samples from drying out. For subculturing, portions of the NNA containing trophozoites/cysts free of fungal and bacterial contamination were screened under a light microscope (with a 10× objective lens), marked, cut with a sterile scalpel, and cultivated onto new plates (Chomicz et al. 2023). Incubation temperature and duration were the same as in primary cultures (Fatemi et al. 2023). Amoebae culture plates were examined using morphological criteria of their cysts and trophozoites based on the Page keys (Page 1988).

FLA were harvested following previously established protocols (Mahdavi et al. 2024). Briefly, plates were washed with 1 mL of sterile phosphate-buffered saline (PBS; pH = 7.2), and the amoebae were concentrated by centrifugation at 2,000 rpm for 5 min. After that, the supernatant was discarded, and DNA was extracted from the remaining pellet using a total nucleic acid extraction kit (Favorgen Biotech, Taiwan). Polymerase chain reaction (PCR) amplification was conducted using specific primers including JDP1 (5′-GGCCCAGATCGTTTACCGTGAA-3′) and JDP2 (5′-TCTCACAAGCTGCTAGGGGATA-3′) for Acanthamoeba spp. (Schroeder et al. 2001), ITS1 (5′-GAACCTGCGTAGGGATCATTT-3′) and ITS2 (5′-TTTCTTTTCCTCCCCTTATTA-3′) for Vahlkampfiidae (Pélandakis & Pernin 2002), Hv1227(5′-TTACGAGGTCAGGACACTGT-3′), and Hv1728R (5′-GACCATCCGGAGTTCTCG-3′) for V. vermiformis (Kuiper et al. 2006), and 5′-Balspec16S (5′-CGCATGTATGAAGAAGACCA-3′) and 3′-Balspec16S (5′-TTACCTATATAATTGTCGATACCA-3′) for B. mandrillaris (Booton et al. 2003). The PCRs were performed in a total volume of 15 μL, which included 7.5 μL of 2× red master mix (Ampliqon, Denmark), 10 ρmol of each primer, DNA (at least 10 ng), and distilled water. The thermal cycling profile was 94 °C for 3 min, followed by 35 repeated cycles, including 94 °C for 35 s, 55–56 °C for 1 min, and 72 °C for 1 min. The final extension cycle was performed at 72 °C for 5 min (Mahdavi et al. 2024). A sequenced isolate for each FLA was employed as a positive control (Acc. Nos. PP659719.1 for Acanthamoeba spp.; MF462211.1 for V. vermiformis, KR908791 for B. mandrillaris, and MN933852.1 for Naegleria sp.). The amplification of each target was confirmed by visualizing the PCR products on a 1.3% agarose gel stained with Safe Stain, which was then viewed under UV light. All PCR products were subsequently sequenced using an ABI 3130 sequencer (CA, USA).

Sequences were edited and visualized using Chromas version 2.6.6 and aligned with ClustalW, which is integrated into BioEdit version 7.0.9. Ambiguous sites were verified against available sequences in the GenBank database. The edited sequences were compared to the GenBank database using the Basic Local Alignment Search Tool (BLAST) to determine their genotypes. All sequences were submitted to GenBank under the following accession numbers: PQ056824–PQ056838. The aligned sequences were screened based on a consensus sequence to identify potential gaps, and the sequences were trimmed.

The Bayesian Information Criterion was employed based on the model nucleotide substitution to choose the best phylogenetic tree. The maximum-likelihood algorithm and Tamura 3-parameter option incorporated in the molecular evolution genetic analysis (MEGA) X software (Kumar et al. 2018) were employed. Bootstrap with 1,000 replications was also executed to evaluate statistical support for distance.

To elucidate the genetic correlation between the sequences of this study and those previously isolated from various sources, including surface water, soil, AK patients, hospital wastewater, and vegetables, DnaSP v5 (Librado & Rozas 2009) was employed.

Haplotype diversity (Hd), segregation sites (S), nucleotide diversity (π), gene flow, and Tajima's D test were calculated for FLA-positive samples.

Network analysis using PopART was performed to explore potential infection sources and assess their distribution and adaptability across various ecological niches (Leigh & Bryant 2015). In this order, the transitive/tree-based consistency score (TCS) network was constructed using a parsimony-based analysis of the aligned sequences, employing the TCS method (Clement et al. 2000) to visualize the inter- and intra-species genetic relationships. This approach allows for a clear understanding of the genetic connections between isolates, highlighting their evolutionary pathways and providing insights into species diversity and potential transmission dynamics. In this study, we examined the genetic diversity and relationships among the genotypes of Acanthamoeba spp. isolates from different sources.

There were two mixed plates in FLA-positive samples, showing co-occurrences of Acanthamoeba spp. and Vahlkampfiidae in one sample and Acanthamoeba spp. and Vermamoeba sp. in the other sample. There was no microscopical evidence for B. mandrillaris in plates. The contamination of rivers was 33% (4/12) in Jajrood, 25% (3/12) in Shadchay, 58% (7/12) in Kan, 25% (3/12) in Darakeh, and 8.3% (1/12) in Farahzad. Most positive samples were from samples collected during spring (three, three, and one positive samples for March, April, and May, respectively) and summer (two and four samples for July and August, respectively); however, the association between season and FLA occurrence was not significant (P-value = 0.083). In addition, there was no significant association between the presence of FLA and water temperature (P-value = 0.207), environment temperature (P-value = 0.391), pH (P-value = 0.974), and solar radiation (W/m²) (P-value = 0.147) (Tables 1 and 2) (Figure 2).

All microscopic positive samples were further analyzed using a PCR assay. From 18 samples that were microscopically identified as FLA, 15 samples were molecularly positive. In this regard, from 17 morphological Acanthamoeba spp.-positive samples, an approximately 450-bp fragment, specific to Acanthamoeba spp., was successfully amplified in 15 plates. Despite several attempts, three samples including two microscopically Acanthamoeba spp.-positive and one Vermamoeba spp.-positive samples were not successfully amplified and sequenced due to the high bacterial and fungal contaminations (Table 1).

Four haplotypes were detected in the genotype T4, which was more than other genotypes. The results of DnaSP analysis showed that the π index for the genotype T4 (0.03404) was higher than that for T11 (0.01419). In addition, the number of segregating sites and mutations indicates that the distribution of single-nucleotide polymorphisms in the T4 genotype is higher than in the other two genotypes, supporting the greater number of haplotypes in this genotype. Interestingly, all sequences of the genotype T3 were similar, and the π index was zero. The results of neutrality tests indicated positive (0.49618) and negative (−1.19955) Tajima's D values for the genotypes T4 and T11, respectively. Statistical analysis revealed that the value of Tajima's D was insignificant for both genotypes (Table 3). Our results revealed the gene flow between the T4 genotype isolates derived from the river in Tehran and T4 sequences retrieved from AK patients (Hajialilo et al. 2016), household biofilm (Norouzi et al. 2021), and hospital wastewater samples (unpublished data) from Tehran (Table 4). Except for two Acanthamoeba T4 populations, hot spring and river water, in which the F_st index was more than 0.33, indicating the lack of gene flow and genetic relationship, the comparison of the index in other populations suggests close genetic relationships between the T4 genotypes. This result suggests the probability of high genetic diversity in Acanthamoeba T4 genotypes living in hot springs to increase their chance of enduring high-temperature environments (Table 4).

In this study, the presence of FLA in selected recreational rivers of Tehran, the capital of Iran, was identified in 18/60 (30%), in which Acanthamoeba spp. was the dominant FLA. In this study, two Vermamoeba sp. and one Vahlkampfiidae were microscopically identified. Acanthamoeba spp. has been reported as the major FLA in most studies, not only in environmental samples but also in clinical settings (Fonseca et al. 2020; Andalib et al. 2022; Borella da Silva et al. 2023; Mahdavi et al. 2024). The high prevalence of Acanthamoeba spp. in different samples seems to be linked to the ability of this protozoan to remain viable in diverse environments such as water, air, soil, and household biofilm (Khan 2006; Salazar-Ardiles et al. 2022). However, the variation in the prevalence of Acanthamoeba spp. may be attributed to the detection methods, environmental and water temperatures, and weather conditions (Chaúque et al. 2023; Menacho et al. 2024). In addition, domestic and industrial sewage and stagnation of seasonal basins can lead to the formation of biofilm, which increases the prevalence of FLA (Pinto et al. 2021; Masangkay et al. 2022).

The prevalence result of this study is in harmony with other studies conducted in Iran. Javanmard et al. (2022) reported the presence of FLA a few miles away from this study location in the surface waters of Alborz province, where their prevalence was lower than that found in this study. In 2017, a survey of surface water and hot springs in Semnan province reported a prevalence of 20% for Acanthamoeba spp. (Javanmard et al. 2017). The results of this study are consistent with two studies, one in Uganda investigating environmental samples including river water, canal water, and tap water from the Queen Elizabeth Protected Area indicating a prevalence rate of 33% (Sente et al. 2016) and one examining water samples of coastal lagoons in Australia (Rayamajhee et al. 2023) reporting a prevalence rate of 38.3%. However, the prevalence of Acanthamoeba spp. in the current study was lower than the 80% reported in river water in the Philippines, which may be attributed to the smaller sample size (Garrido et al. 2023).

Although a wide range of FLA are reported from environmental samples, such as surface waters, Acanthamoeba spp. is the most common FLA in clinical samples. The prevalence of Acanthamoeba spp. in clinical samples in Tehran has also been around in our study (Hajialilo et al. 2016; Memari et al. 2016). Memari et al. (2016) indicated that the prevalence rate of Acanthamoeba spp. was 13.4% in the oral mucosa of immunocompromised patients. The prevalence of AK in Tehran hospitals was reported to be 13% (Hajialilo et al. 2016). Arab-Mazar et al. (2021) reported that 7.5% of heart transplant patients in Tehran were positive for Acanthamoeba spp. in their mucous membranes. The presence of Acanthamoeba spp. in water resources and clinical samples in Tehran with similar prevalence highlights the risk of the transmission of the protozoan from the environment to humans. This risk is strongly supported by studies performed by Fatemi et al. (2023), which showed a high prevalence of FLA in fresh vegetables collected from municipal markets in Tehran. Interestingly, the source of the fresh vegetables sold in Tehran is farmlands that are naturally irrigated by branches of rivers, as investigated in the current study.

Sunlight, through solar radiation (W/m²), has long been regarded as a potent agent for biological control, often employed to mitigate the impact of various microorganisms. However, its efficacy against certain resilient organisms, particularly the cysts of Acanthamoeba spp., has remained an interesting topic of research (Chaúque et al. 2023). The current study identified a notable increase in positive samples of Acanthamoeba spp. during the warm months with heightened levels of sunlight, although it was not statistically significant. No statistical relationship was observed between ambient temperature and pH in this study. However, studies show that the optimal temperature around 30 °C and pH, and neutral conditions provide the best performance for the growth of Acanthamoeba spp. (Lakhundi et al. 2014).

Based on the results, the identified genotypes of Acanthamoeba included T4, T11, T3, and T5. These genotypes have been characterized in Tehran's environmental and clinical samples (Nazar et al. 2011; Behnia et al. 2017; Norouzi et al. 2021; Fatemi et al. 2023). For instance, Pazoki et al. (2020) successfully isolated and characterized the genotypes T2, T4, T5, and T11 from farmlands and recreational soils in the southern regions of Tehran. Furthermore, the T4 and T5 genotypes were found in fresh vegetables sold in nearby municipal stores (Fatemi et al. 2023). These vegetables are primarily sourced from farmlands that are partially irrigated with river water (Pazoki et al. 2020; Fatemi et al. 2023). This correlation highlights the high prevalence of clinically significant Acanthamoeba genotypes in river water and the close association of these water resources with human activities, emphasizing the environment's role in transmitting FLA to humans.

Among these genotypes, the T4 strain of Acanthamoeba spp. stands out as one of the most pathogenic. It is notably linked to severe infections such as AK, which can result in significant morbidity (Diehl et al. 2021). Given its established pathogenic potential, studying the T4 genotype is crucial for enhancing our understanding of its distribution and the implications for public health.

Haplotype diversity (Hd) is a key measure in population genetics that provides insights into a species' evolutionary history. It helps researchers to trace lineages, understand migration patterns, and assess the geographical distribution of specific haplotypes. High Hd often indicates a robust genetic pool, which enhances a population's adaptability to environmental changes and resilience against diseases (Spotin et al. 2017). In this study, the T4 genotype exhibited a high Hd of 0.900, surpassing the previously reported value of 0.636 for T4 genotypes isolated from AK patients (Spotin et al. 2017). This suggests that the T4 populations are experiencing complex dynamics driven by expansion, selection, and environmental adaptability. The current findings imply that changes in Hd may reflect ecological shifts or emerging public health threats. High genetic diversity in Acanthamoeba T4 could indicate varying virulence or resistance traits and signal potential risks for emerging infections. Understanding this diversity is crucial for developing effective public health strategies to mitigate infection risks related to these amoebae.

Tajima's D statistic is used to assess the genetic diversity within a population, providing insights into its evolutionary dynamics. A positive Tajima's D value indicates potential population expansion or balancing selection (Tajima 1989). As a result, the T4 genotype displayed a positive Tajima's D value of 0.49618, suggesting intriguing population dynamics. This may indicate that the population is either expanding or experiencing balancing selection, which can have significant implications for its evolutionary adaptations.

The phylogenetic analysis supports the idea of a transmission cycle for FLA, particularly Acanthamoeba spp., between environmental sources and humans in Tehran province. This aligns with the One Health approach, which promotes an integrated perspective on health that encompasses environmental and human health. The One Health approach encourages comprehensive surveillance and research considering various factors influencing FLA transmissions, such as environmental changes and human behaviors. By fostering collaboration across disciplines, we can enhance our understanding of Acanthamoeba epidemiology and develop effective prevention strategies to combat related infections.

In this study, 15 of 60 river water samples tested positive for Acanthamoeba spp. by PCR. Molecular analysis revealed the presence of genotypes T3, T4, T5, and T11 in the positive samples. The T4 haplotypes in this study were closely related to those derived from AK patients, hospital wastewater, and soil isolates. The findings suggest a clear association between environmental and clinical isolates of Acanthamoeba spp., particularly where there is high human activity and close contact between humans and water resources. Although microbial screening of water resources with high human activities is performed, surface and river waters are usually overlooked. Therefore, the risk of the transmission of FLA through river waters is still high. Given the high level of human activity at the sampling sites and the use of this water for recreational and agricultural purposes, routine monitoring is highly recommended. In addition, establishing warning signs along rivers with high human activity, such as buildings, farming, and restaurants, is crucial.

All experimental protocols were in accordance with the ethical principles and the national norms and standards for conducting Medical Research in Iran. The study was performed in accordance with the relevant guidelines and declaration. The current study was approved by the Tehran University of Medical Sciences (IR.TUMS.SPH.REC.1402.117).

All authors declare that they have seen and approved the submitted version of this manuscript.

This study was financially supported by the School of Public Health, Tehran University of Medical Sciences (No. 68985).

E.K. and H.M. were responsible for the conceptualization and study design. E.J., H.M.R., and M.A. conducted the investigation and formal analysis. E.J. and M.M. handled software development, while validation and data curation were carried out by H.M., E.K., and M.R. The original writing and editing were completed by E.J., H.M., and E.K. All authors read and approved the final version of the manuscript.

The authors thank all members of the Foodborne and Waterborne Diseases Research Center for their support.

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