First report of Acanthamoeba spp. isolation in a recreational hot spring in the Philippines

Hot springs are popular tourist destinations in several parts of the world (Kusdibyo 2021). These sites' natural waters offer recreational and medicinal benefits (Valeriani et al. 2018; Vaidya & Nakarmi 2020). Several studies have proven that the warm waters from these springs are therapeutic to several musculoskeletal problems (Ranjit 2022). Due to this, hot springs are among the tourist sites in the world categorized as wellness tourist sites (Medai et al. 2022). Recent reports show that the number of tourists who frequent these sites has grown astronomically in the last eight years (Yeung & Johnston 2018). Japan, for instance, has been regarded as having the largest market in terms of its hot springs (Suzuki & Asanuma 2021).

Aside from their recreational and medicinal benefits, hot springs serve as an excellent environmental matrix for several microorganisms. Organisms considered thermophilic can thrive in the high temperature of its waters. In concluded studies, several bacteria, viruses, and eukaryotic organisms have been isolated from the waters of hot springs (Massello et al. 2020). Although most of these are nonpathogenic, a few thriving in hot springs would tell otherwise (Sadeepa et al. 2022). Among those considered clinically significant microorganisms isolated from hot springs are free-living amoebae (FLA) (Fabros et al. 2021). The Centers for Disease Control and Prevention identify four genera of FLA of health importance; these are Acanthamoeba spp., Balamuthia spp., Naegleria spp., and Sappinia spp. (CDC 2019). These four genera can induce a meningitis-like infection in mammalian hosts, leading to fatal outcomes due to a lack of established therapeutic and diagnostic protocols (Milanez et al. 2022). Among the medically significant FLAs, Acanthamoeba spp. is the most occurring FLA in terrestrial and aquatic matrices (Garrido et al. 2023). Its high adaptability to different environments makes it a focus of several surveillance studies (Ardiles et al. 2022; Flores et al. 2023). Swimming in FLA-contaminated waters may consequently lead to conditions such as fatal Granulomatous Amoebic Encephalitis or the non-fatal eye infection Acanthamoeba keratitis (AK) (Mungroo et al. 2022).

Hot springs in the Philippines are popular tourist destinations, but they lack comprehensive surveillance and regulation, posing a challenge for local and national administrators (Jago-on et al. 2017). This study addresses this gap by focusing on the molecular identification of Acanthamoebae spp. in a hot spring resort in the country. To our knowledge, this is the first surveillance of FLAs in a hot spring in the Philippines, underscoring the need for such research.

A total of 40 surface water samples were collected from the five identified collection sites (eight water samples for each site). Collection sites are designated 200 m apart to cover the entire study site. Surface water samples were collected approximately 10–20 cm from the surface. Water samples were placed in sterile collection bottles and stored at room temperature until processed in the laboratory. Two-hundred fifty mL of the collected surface water samples were filtered through a 1.2 μm pore size, 47 mm diameter glass microfiber filter (Whatman™, United Kingdom) using a vacuum pump. The filter paper was placed top down on the previously prepared non-nutrient agar (NNA) lawned with Escherichia coli, incubated at 30 °C, and microscopically checked daily for amoebic growth for 14 days using a regular compound microscope (Olympus CX23) under 400× magnification. Culture plates with amoebic growth were subcultured to obtain homogenous growth (Milanez et al. 2019).

Amoebic cysts and trophozoites were harvested from positive subcultured plates by flooding the agar surface with PAGE amoeba saline stored at 2–8 °C to detach cells on the surface of the agar plate. The surface was gently scraped with a sterile scalpel blade, and approximately 800 μL of fluid suspension was then aspirated and transferred to microcentrifuge tubes. DNAs were extracted using the DNeasy Power Soil Kit (Qiagen™, Netherlands) following the manufacturer's protocol. Extracted DNAs are stored at 4 °C or used as a template for polymerase chain reaction (PCR) amplification immediately. DNAs were made to react to PCR (BioRad T100 Thermal Cycler©) using primer sets JDP1 5′GGCCCAGATCGTTTACCGTGAA-3′ and JDP2 5′TCTCACAAGCTGCTAGGGAGTCA-3′ for cells that resemble Acanthamoebae spp. PCR conditions were set as follows: 95 °C for 7 min for initial denaturation, 40 cycles of denaturation at 95 °C for 1 min, the annealing temperature of 55 °C for 1 min, extension at 72 °C for 2 min, and a final extension of 72 °C for 15 min (Booton et al. 2005). DNA was visualized with 1.5% agarose gel stained with Gel Red^® (Biotium, United States). Amplicons were sent to a commercial sequencing company (1st Base, Singapore) for further sequencing. Sequences were aligned using ClustalW of BioEdit with careful visual consideration of gaps and ambiguous sequences.

The positive isolates were subjected to a thermotolerance assay patterned to a previously established protocol with a few modifications (Padua et al. 2023). Briefly, 5 μL of cystic suspension was inoculated at the centre of freshly prepared NNA lawned with E. coli plates and incubated at varying temperatures of 25, 30, and 40 °C. Amoebic growth is microscopically checked after 48 h with emphasis on the migration of trophozoites from the point of inoculation.

The isolation of Acanthamoeba spp. in hot springs is not surprising. As previously mentioned, the thermophilic characteristic of this FLA enables it to survive even in the harshest of environments (Celis et al. 2023). However, it is important to consider the occurrence of these organisms in such sites. Recreational sites like hot springs are considered an anthropogenic and biological activity nexus. For this reason, the chance of accidental encounters increases as the organisms proliferate in the waters.

Conversely, activities conducted in these reservoirs lead to exposure of eyes and nasal passages that serve as potential entry points for these organisms. This scenario has been observed in several cases concerning host exposure to an environment containing FLA (Aparicio et al. 2021). In this study, the occurrence of FLAs in the hot spring may have been the cause of several factors. Temperature, soil runoff, and the potential abundance of microorganisms in the area are essential factors to consider. FLAs are thermophilic organisms, and the natural temperate waters of the spring enable FLAs to thrive. It has been mentioned that the spring is in direct contact with the soil. This enables terrestrial organisms, including FLAs, to be incorporated into the waters. It does not only introduce terrestrial FLAs into the water but, more importantly, microorganisms that serve as food sources, enabling FLAs to thrive and feed.

The occurrence of Acanthamoeba genotype T7 in a recreational freshwater environment may pose a public health concern. The pathogenic capacity of this FLA has been reported in recent studies, and it can cause AK (Eldin et al. 2019). Swimming and other water-related activities performed in the hot spring, which eventually expose the eye to FLA-contaminated water, are considered high risk. Therefore, it is necessary to consider the regular surveillance and maintenance of recreational hot springs frequented by local and foreign tourists. This will, consequently, prevent possible future infections from these environmental sources. In this study's case of the Acanthamoeba T20 isolate, although no clinical cases in humans have been reported, it is essential to note that this isolate caused a fatal avian infection (Fuerst et al. 2015). Considering the resiliency of Acanthamoeba spp. and its capacity to cause a spectrum of human diseases, dismissing this isolate's capacity to induce human infection would be detrimental.

Furthermore, our thermotolerance testing suggested a high possibility for this isolate to be pathogenic. This further validates the existence of variant strains within Acanthamoeba groups (Milanez et al. 2020). In our study, our genotype T7 isolate exhibited the capacity to increase at 40 °C, suggesting it can be potentially pathogenic.

Finally, hot springs are recreational sites that should be monitored and maintained. The reason may have been to preserve its natural esthetic beauty that appeals to tourists. Although this is the case, it is vital to consider the different biological and microbiological organisms that can potentially thrive. The current climatic changes, such as increased rainfall that causes soil runoff, flooding, and rising temperature during the summer months, contributing to abnormal increases in water temperature, all affect the proliferation of these potentially pathogenic organisms. With that said, it is paramount to consider surveillance and monitoring of natural hot springs, especially those used for recreational purposes, to avoid future cases of FLA infection.

Natural hot springs are ideal places to retreat and escape the busy work in the city. They offer relaxation to people and, from a biological standpoint, are considered home to several microorganisms. This study provides the first evidence of FLA isolation in a natural hot spring in the Philippines. The presence of Acanthamoeba spp. in recreational freshwater sources does not equate to immediate infection but, rather, a risk and a potential source of future infection. It suggests, however, to conduct regular monitoring, maintenance, and surveillance in natural hot springs, especially those used for recreational purposes. Local government involvement is highly recommended to provide policies and regular trucking of private resorts in compliance.

The authors express their most profound and sincerest gratitude to the Department of Medical Technology, Faculty of Pharmacy, University of Santo Tomas, for providing technical support.

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

The authors declare there is no conflict.