Isolates of Acanthamoeba species in the marine environment in the Philippines

Free-living amoebae (FLA) are important emerging pathogens in modern times (Milanez et al. 2022). Their ability to induce fatal outcomes in human health has made this group one of the significant public health concerns. The World Health Organization (WHO) has identified important pathogens under four genera: Naegleria spp., Balamuthia spp., Sappina spp., and Acanthamoeba spp. (WHO 2003). Among the four genera, the latter is considered more ubiquitous and of greater public health concern than the others (CDC 2019). This may be due to its ability to induce fatal and non-fatal infections in a spectrum of mammalian hosts. This genus can cause a fatal central nervous system infection and a non-fatal eye infection. Although a proven diagnostic protocol and therapeutic regimen are available for treating Acanthamoeba keratitis, there appears to be none for fatal granulomatous amoebic encephalitis. Due to their clinical importance and ability to act as Trojan horses (Mungroo et al. 2021), the isolation of Acanthamoeba spp. in different environmental sources has recently become the focus of several studies.

Acanthamoeba spp. have been isolated from aquatic sources such as rivers (Garrido et al. 2023), lakes (Milanez et al. 2020; Halim et al. 2023; Karimi et al. 2023), volcanic mud springs (Celis et al. 2023), and groundwater (Ramirez et al. 2006; Padua et al. 2023). Researchers have also documented terrestrial isolation in both soil (Abedi et al. 2021) and permafrost samples (Malavin & Shmakova 2020). The isolation of Acanthamoeba spp. is not limited to environmental sources; recent biological isolation has been reported from bat guano (Mulec et al. 2016) and fish organs (Im & Shin 2003; Milanez et al. 2024). Moreover, the clinical isolation of Acanthamoeba spp. has been confirmed from urine samples of patients with recurring urinary tract infection (UTI) infections (Santos et al. 2009; Saberi et al. 2021). The increasing evidence of the ability of this FLA to be present in almost all types of samples has aroused many researchers' interest in conducting further investigations into possible unexplored FLA habitats. These unlikely habitats include marine environments, but recent reports of its isolation in marine waters would say otherwise (Latifi et al. 2020; Sousa-Ramos et al. 2022).

This warrants further investigation into the possible presence of FLAs, particularly Acanthamoeba spp., in shorelines in the Philippines in Mariveles, Batan, and Lingayen Gulf.

The selection of sites is based on accessibility, the presence of community, and the presence of aquaculture. The collection sites in Mariveles, Bataan MB1–MB3 shorelines are all situated immediately near communities where people use the shoreline for recreation and fishing. It is important to note that MB3 is an estuary where dead remains of animals are seen. Site MB4 is a previous port converted into a shipyard, while MB5 is situated in a private resort with several jellyfish present at the time of collection. All collection sites in Lingayen Gulf are recreational sites frequently visited by local and foreign tourists. Lingayen Gulf is located on the Northwestern coast of Luzon, Philippines, and is a semicircular body of water teeming with life connected to the South China Sea.

A total of 80 surface water samples were collected from the two different shorelines (five identified sites in Mariveles, Bataan, and four sites in Lingayen Gulf). Four water samples were collected 50 m from the shoreline for each site, while six samples were collected 100 m from the shore. Surface water samples were placed in sterile collection bottles and stored at room temperature until being processed in the laboratory. Two-hundred and fifty milliliters of the collected surface water samples were filtered through a 1.2 μm pore size, a 47 mm diameter glass microfiber filter (Whatman™, UK), and using a vacuum pump. The filter paper was placed upside down on the previously prepared non-nutrient agar (NNA) that was lawned with Escherichia coli and incubated at 30 °C. It was then microscopically checked daily for amoebic growth for 14 days using a regular compound microscope (Olympus CX23) at 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 PowerWater Kit (Qiagen™, Netherlands) following the manufacturer's protocol. Extracted DNAs were stored at 4 °C or used as a template for polymerase chain reaction (PCR) amplification immediately. DNAs were subjected to a 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, an 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 a 1.5% agarose gel stained with Gel Red^® (Biotium, USA). 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 thermotolerance assay on sequenced isolates was performed following the previously established protocol (Padua et al. 2023) with a few modifications. Briefly, 100 μL of cystic stage suspension of isolates was cultured at the center of freshly prepared NNA plates lawned with live E. coli and incubated at varying temperatures, i.e. 25, 30, and 40 °C. Amoebic growth was observed for 48 h, emphasizing the outward migration of trophozoites from the point of inoculation.

Acanthamoeba spp. are the most resilient members of the FLA group. This ability to adapt to a wide range of environmental habitats has made them the most common FLA isolated in the environment. Their ubiquity consequently results in higher human encounters and documented outbreaks, as several recent studies show (Carnt et al. 2018). The occurrence of saline-tolerant FLAs demonstrates not only the ability of this organism to acclimatize to environments it usually does not inhabit but also the increase in environmental territory in which it can thrive. Such adaptive characteristics may mean the potential ability of some species within this clade to invade extreme anatomical parts other than the brain and cornea. Conversely, it has been proven that genotype T4 has been isolated in the urinary tract in two reports in recent years (Santos et al. 2009; Saberi et al. 2021). Marine water isolates of Acanthamoebae spp. offer the basis of the high saline tolerance of some species in this genus and their ability to invade, thrive, and cause damage to mammalian hosts. The presence of Acanthamoeba in the sampling sites of this study may be attributed to three critical factors. Firstly, as previously mentioned, Manila Bay is considered under rehabilitation due to damage caused by biological and industrial wastes dumped from surrounding coastal communities. This translates to the proliferation of bacteria, which consequently serve as food sources of FLAs. This may exacerbate the proliferation of Acanthamoeba in the sampling sites. Second, different estuaries coming from the rivers are left unchecked and, like the bay itself, are considered heavily polluted from biological and industrial waste. Acanthamoeba-containing brackish water eventually drains into the bay, allowing the former to adapt to the environment. Third, expanding human settlement surrounding the bay further increases anthropogenic activity and potential soil runoff into the bay's waters. As human activity increased, soil containing Acanthamoeba may have been incorporated into the bay's waters. This was observed from our previous investigation concerning the occurrence of FLAs in certain parts of a lake where communities are present (Milanez et al. 2019). Although the Lingayen Gulf shoreline may not have the same potential risks on why Acanthamoeba isolates, like A. culbertsoni and genotype T4, are known to cause keratitis in mammalian hosts (Diehl et al. 2021). Our isolation of A. culbertsoni in this study agrees with previous results from other isolations of FLAs in marine environments belonging to the T4 genotype classification (Rayamajhee et al. 2023). However, unlike previous studies, our isolates showed no pathogenic attributes when subjected to thermotolerance tests. Although this is the case, determining the pathogenic ability of environmental isolates of Acanthamoeba should be supported by other in vivo testing as argued by other studies (Sousa-Ramos et al. 2022). Conversely, although our isolates in this study failed to grow beyond 30 °C, their potential ability to induce pathogenic effects through various means should be taken into consideration.

Over the years, the anthropogenic activities in Manila Bay have ranged from port operations and urban expansion to pollution discharge and have substantially altered its natural dynamics. Research suggests that urban and industrial pollution has primarily affected water quality deterioration, which has directly endangered the health of aquatic animals, ultimately leading to a decline in marine resource production and marine habitats. This is evident in commonly consumed and growing bivalve species such as oysters (Crassostrea iredalei) and green mussels (Perna viridis), in which enteric pathogens such as Cryptosporidium have been found in bivalve samples collected in Manila Bay, posing a significant health risk to the community (Pagoso & Rivera 2017). A common cause of pollution within Manila Bay is the presence of heavy metals such as lead, cadmium, and chromium, which have grown significantly because of industrial waste, sewage runoff, and agricultural waste detected in the water sample along with the presence of polycyclic aromatic hydrocarbons, benzotriazole ultraviolet stabilizers, and plasticizers (Montojo et al. 2021). Consequently, urban pollution contributed to the deterioration of marine life to support the increased population of aquatic microorganisms. It is suggested that the surface water temperature, average pH, and dissolved oxygen level of the water are generally ambient and are considered optimal for the survival and proliferation of microorganisms and bacterial communities. While Manila Bay is considered a possible environment for microbial proliferation, pollution has greatly contributed to the increase in microbial growth in the area. Evidence of microbial growth can be seen in the floating plastic debris collected in Manila Bay, which shows a variety of microorganisms attached to the plastic waste (Cruz & Shimozono 2021). Moreover, due to other human activities, such as ballasting operations of ships, Manila Bay may support a diverse array of microorganisms, including bacteria, viruses, protists, and fungi. In 2019, the Department of Environment and Natural Resources (DENR) issued inspection notices to businesses in the area after determining the composition of the bay's wastewater. Results showed untreated hospitals, animals, detergents, and food waste. An estimated 330 million MPN fecal coliforms were isolated from the river basin alone (Rafales 2019). As such, rehabilitation programs surrounding pollution were launched to restore the bay's sustainability. Although parasitic protozoans have been documented in the bay, there is currently a lack of information on potential FLA occurrences in the area.

Similarly, Lingayen Gulf's shoreline exhibits remarkable diversity, encompassing sandy beaches, mudflats, and mangrove ecosystems. These dynamic interfaces between land and water harbor a rich tapestry of life, and the composition and structure of these systems may contribute to the occurrence of FLAs in the area. Such factors include sediment composition, tidal patterns, and nutrient fluxes, which collectively influence the composition and dynamics of microbial communities, which consequently influence the presence of FLAs. Considering these characteristics offers valuable insights into the potential niches and ecological roles that support the presence and distribution of FLAs within the Gulf. Furthermore, the Gulf of Lingayen is considered one of the primary tropical destinations in the province of Pangasinan for nature tourism. It attracts a significant number of travelers and tourists every year. Despite all this, the geographic location remains a research gap. It needs to be more researched due to limited sources, justifying that this field of study in the Philippines needs further research. Addressing this research gap is essential for academic purposes, as well as for informing sustainable management practices and policies and ensuring the long-term conservation of this invaluable marine ecosystem in the Philippines. From a public health perspective, occurrences of potentially pathogenic Acanthamoeba spp. in waters used for recreation and livelihood suggest health concerns to the public. It has been observed that people, particularly children, are using the bay to swim during the summer months to cool themselves from the intense heat. This activity increases the chance of accidental encounters while engaging in such water activity. It should be noted that among the FLAs, Acanthamoeba spp. has several points of entry to induce pathogenic effects. It can enter the system through the nasal passages, eye exposure to contaminated water, or the skin via open wounds. With its multiple transmission routes, the chance of contracting infection increases upon exposure to contaminated water sources. The limited reports concerning the presence of this FLA in marine waters, the low undocumented cases concerning the infection of FLAs in the country, and the absence of policies in regulation and monitoring of coastal waters for FLAs are enough reasons to seek and establish new policy guidelines in its monitoring and surveillance.

Acanthamoeba spp. is considered one of the emerging pathogens in recent years. The lack of established surveillance and monitoring of this FLA in different environmental matrices used for recreation and livelihood requires immediate attention. The isolation of Acanthamoeba spp. in a marine environment reflects a decline in water quality. Further surveillance of similar environments is highly recommended to establish other FLAs that may be present. Potential risk factors such as waste disposal, expansion of human settlements to coastal locations, and soil runoffs in these environments should be controlled to mitigate the proliferation of potentially pathogenic strains of FLAs. Consequently, this will reduce the possible future infections that may occur.

The authors thank Mr Robert Li and Mr Jeffrey Estioko for their technical support. Prof. Maria Ruth Pineda-Cortel, Ph.D., Ms Jezlaine Balagtas, Ms Julian Antonio, and the University of Santo Tomas, Faculty of Pharmacy, Department of Medical Technology, for their technical assistance.

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

The authors declare there is no conflict.