Detection of Acanthamoeba spp. in two major water reservoirs in the Philippines

Water reservoirs are important manmade structures providing water security to deliver clean and safe water for drinking and other purposes to the community (Ðorđević & Dasic 2011). The World Health Organization (WHO) estimates that around 3.4 million people die every year due to water-related diseases and it is becoming the leading cause of morbidity and mortality in the world (Berman 2009). The genus Acanthamoeba is considered an important pathogen of our time due to its ability to cause opportunistic and non-opportunistic infections in humans (Marciano-Cabral & Cabral 2003). These eukaryotic organisms belong to a group of amoebae known as free living amoebae (FLA) that are widely distributed in the environment as well as in recreational pools, cooling towers, water tanks, tap water systems, dental units, air conditioning equipment and others (Scheid 2018; Di Filippo et al. 2015). Furthermore, FLA have been reported to be isolated from bat guano (Mulec et al. 2016) and in bromeliads (Landell et al. 2013). Acanthamoeba spp. was detected in Brazil's recreational waters, where three genotypes were identified (Caumo & Rott 2011). Acanthamoeba spp. has been isolated in Asia and the Pacific region water resources including thermal hot springs in Taiwan (Kao et al. 2012) and Thailand (Lekkla et al. 2005), as well as water reservoirs in China (Lass et al. 2017) and Japan (Edagawa et al. 2009). There are currently 18 species of Acanthamoeba and these are classified into three groups based on the features of endocyst and ectocyst (Ubelaker 2014). Acanthamoeba spp. are known to cause two major human infections: granulomatous amoebic encephalitis (GAE) which affects the central nervous system and usually is fatal to the host and Acanthamoeba keratitis (AK) that can cause blindness (Khan 2006). Moreover, Acanthamoeba spp. are reported to cause cutaneous infections, especially in immunocompromised patients, which are described as erythematous papules, pustules or linear lesions with erythematous background (Deluoi et al. 1996; Casper et al. 1999). Aside from its virulence to cause infection, Acanthamoeba spp., being a natural phagocyte, is considered a ‘Trojan horse’ which is able to harbour endosymbionts such as viruses, yeasts, protists and bacteria that are pathogenic to humans (Greub & Raoult 2004).

Isolation and characterization studies of Acanthamoeba spp. in the Philippines are limited to clinical samples from contact lenses (Rivera & Adao 2009; Buerano et al. 2014) and nasal swabs (Cruz & Rivera 2014). Point source isolation and identification of Acanthamoeba from main water reservoirs have yet to be studied in the country. Magat and Ipo water reservoirs are considered as the main water reservoirs supplying potable water in the island of Luzon, Philippines. The Magat reservoir contributes to water irrigation for crops, fish production, domestic uses and electricity generation (Elazegui & Combalicer 2004) while the Ipo reservoir, which is linked to the Angat-Umiray-Ipo watersheds, provides 98% of the water needs of the capital Metro Manila (WWF). Being the main sources of water for the public and considering the frequency of human contact, the need to further explore the presence of potential pathogenic FLAs in these environments is crucial to provide awareness and measures that can be taken to prevent human infections. In this study, we isolate and identify possible FLAs present in the two water reservoirs.

A total of 80 water samples were taken from Magat and Ipo water reservoirs (40 water samples in Magat; 40 water samples in Ipo) using purposive sampling between November 2018 and January 2019. Four sampling sites in Ipo watershed were identified, namely, site one – Sitio Bininit (14°53′53″N 121°10′16″E), site two – Sitio Paco (14°53′16″N 121°9′37″E), site three – Sitio Sapang Munti (14°52′13″N 121°9′8″E), site four – Sitio Bitbit (14°54′1″N 121°8′30″E), while the four sampling sites for Magat watersheds were site one – Baligatan (16°48′45.9″N 121°26′47.9″E), site two – Isla Verde (16°48′43.5″N 121°26′34.7″E), site three – Isla Verde (16°48′56.9″N 121°26′38.8″E) and site four – Callao (16°49′09.6″N 121°26′36.3″E). Sample sites were selected based on accessibility, presence of community near the shore as proof of anthroponotic activity or due to the presence of fish farms. Two hundred and fifty mL of water samples were then collected at approximately 10–20 cm depth from the surface and placed in sterile containers (Milanez et al. 2019). The samples were transported to the laboratory at the Department of Medical Technology, Faculty of Pharmacy, University of Santo Tomas, Philippines to identify amoebic growth using culture methods. Samples were transferred to Falcon tubes and pelleted at 3,000 rpm for 15 min. The resulting pellets were evenly lawned to non-nutrient agar (NNA) lawned with Escherichia coli and were incubated at 33 °C (Schuster 2002).

NNA lawned with E. coli culture plates were put in an incubator set at 33 °C and were examined daily for amoebic growth using a regular compound microscope (Nikon Eclipse E100) for 14 consecutive days before being declared negative as per the protocol established by Page (1988). Plates that showed cystic forms and motile trophozoites were further tested and sub-cultured (Milanez et al. 2019). This procedure was done until a homogenous culture of FLA was obtained. Electron microscopy was done using TM300 Hitachi tabletop SEM. Briefly, the surfaces of positive NNA sub-cultured plates were gently scraped to detach cysts and trophozoites and placed onto coverslips. Coverslips were then attached to carbon tape secured on the metal SEM platform for further analysis.

Culture plates with positive trophozoites and cysts as observed by the microscopic analysis were transported to the Department of Medical Technology, Far Eastern University Manila, Philippines for further molecular analysis. Trophozoites and cysts were harvested by flooding the surface of the agar with cold phosphate-buffered saline solution and by gently scraping the agar surface with a sterile scalpel blade and then aspirated (Milanez et al. 2017). Aspirated fluid was then transferred to microcentrifuge tubes and DNA was extracted using Macherey-Nagel DNA extraction kit (NucleoSpin^®) following the manufacturer's protocol. DNA was made to react to polymerase chain reaction using primer set JDP1 5′GGCCCAGATCGTTTACCGTGAA-3′ and JDP2 5′TCTCACAAGCTGCTAGGGAGTCA-3′. PCR conditions were set as follows: 95 °C for 7 min initial denaturation, 40 cycles of denaturation at 95 °C for 1 min, 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. 2004).

To further identify Acanthamoeba genotypes, further sequencing and phylogenetic analyses were performed. In detail, a 1.5% agarose gel stained with ethidium bromide was used to visualize PCR amplicons. Amplicons were sent to a commercial sequencing company (Macrogen, Seoul, South Korea) for further sequencing. Sequences were aligned using ClustalW of BioEdit with careful visual consideration of gaps and ambiguous sequences and deposited with GenBank afterwards. The DNA sequences of isolates IS1B5, IS4B3, MS45, MS42, IS2B1 and IS1B2 obtained from this study were deposited in the GenBank database and are available under accession numbers MK886460, MK909919, MK905437, MK910997, MK911021 and MK886514, respectively.

A total of 12 out of 80 (15%) water samples were positive for amoebic growth obtained from Magat and Ipo watersheds. Specifically, positive growths of FLA were observed from samples in sites one, three and four in Magat reservoir and sites one, two and three of Ipo watersheds. Light microscopic images of both cysts and trophozoites are presented in Figures 1 and 2, respectively. Cystic stages seen under light microscopy are described as irregularly shaped with distinct double wall measuring approximately 10 μm. Trophozoites observed microscopically have a sluggish directional movement with single pseudopodia. The cytoplasm is somewhat granular and some are highly vacuolated. Furthermore, SEM of cystic stages revealed cystic stages with wrinkled surface appearance as shown in Figure 3. All the findings were morphologically compared based on Page's established criteria in respect to form, size and shape (Page 1967) and were initially classified morphologically as Acanthamoeba spp. Enflagellation testing of amoeba isolates was not performed further due to limitations of the methods on the reliability of the test to differentiate amoebae from other groups (De Jonckheere et al. 2001).

Polymerase chain reaction results using JDP1 and JDP2 primers showed band formation between 400 and 500 bp in agarose gel electrophoresis showing the presence of DNA for isolates IS1B5, IS4B3, MS45, MS42, IS2B1 and IS1B2. Isolate M5I3 belonging to Acanthamoeba genotype T5 was used as a positive control. Further sequencing of PCR amplicons and phylogenetic analysis was done and confirmed the presence of Acanthamoeba hatchetti belonging to genotype T11, Acanthamoeba spp. belonging to genotype T3, Acanthamoeba spp. belonging to genotype T4 in Magat and Ipo reservoirs. Table 1 shows the different isolated Acanthamoeba spp. in both Magat and Ipo water reservoirs. Maximum likelihood tree using Tamura 3 parameter model was constructed as suggested as the best tree model using MEGA 7 application (Figure 4). Table 2 shows the reference strains used to construct the tree.

The Magat and Ipo water reservoirs are two important manmade water reservoirs in the Philippines. Magat water reservoir supplies hydroelectric energy, aquatic produce, irrigation and a potable water source to three provinces, namely, Nueva Vizcaya, Quirino and Isabela (Baleta & Bolaños 2016) while Ipo water reservoir is a part of a larger system called the Angat–Ipo–La Mesa water system that directly supplies water to treatment facilities which are then supplied directly to Metro Manila (Masangkay et al. 2014). This study has confirmed the presence and the biodiversity of Acanthamoeba spp. in two major water reservoirs in the Republic of the Philippines, namely, Magat and Ipo water reservoirs, and to the best of our knowledge, is the first report in the country. The presence of FLAs, specifically Acanthamoeba spp., in these two major water reservoirs, is a clear indication of the high risk that these FLAs can be incorporated into the water being supplied to water treatment facilities and in return to households as tap water, increasing the risk of potential transmission to humans.

The ability of Acanthamoeba spp. to graze with a number of potentially pathogenic bacteria such as Legionella and Mycobacteria (Greub & Raoult 2004) and to safely co-exist as endosymbionts within them suggests that there is a possibility that these microorganisms may escape disinfection during the treatment process (Berry et al. 2010). More importantly, the two water reservoirs directly supply tap water to a very large population in the country, thus, the presence of these FLAs in the water may be considered as a public health issue. The presence of genotypes T3, T4 and T11 in the two water reservoirs suggests a high possibility of transmitting Acanthamoeba keratitis (AK) to people who use tap water from faucets to wash their faces, considering that these genotypes are most often associated with AK, as suggested by some studies (Ledee et al. 1996; Booton et al. 2005). This has been proven to be true with a relatively rare case of AK involving a non-contact lens wearer in the Philippines with a history of using tap water to wash his face before acquiring the condition (Buerano et al. 2014).

Finally, genotypes T3 and T4 are considered the most common genotypes isolated in the environment due to the high resistance to chlorine of their cystic stages whereas genotype T11 is considered susceptible (Shoff et al. 2008). The isolation of Acanthamoeba genotype T11 in Magat water reservoir suggests low levels of chlorination as do reports from drinking waters of hospitals in Iran (Bagheri et al. 2010), although the exact measurement of chlorine was not carried out in this study. Chlorination is considered as an initial step in water disinfection due to its ability to inactivate a number of pathogenic microorganisms (Environmental Protection Agency 2011).

This study has shown the importance of point source isolation of potential pathogenic organisms such as Acanthamoeba spp. Further, images obtained from SEM analysis have provided clear differences of cyst size and morphology of the four different genotypes isolated from two water reservoirs in the Philippines. The detection of potentially pathogenic organisms in the environment, particularly in water reservoirs, in which human activities such as swimming and use of water for other purposes like bathing are likely to occur, will further provide awareness to the community of their presence, thus avoiding severe or even fatal health conditions.

The results of this study produce the first description through scanning electron microscopy and molecular techniques of Acanthamoeba isolates in water reservoirs in the Philippines. The presence of Acanthamoeba spp. that can cause conditions such as GAE and AK in water reservoirs increases the chances of human contact, since water coming from these environmental structures is directly provided as water sources for different human activities, which include bathing, washing and, to some extent, use as potable water. Not only is their the chance of acquiring FLAs, but also the infection of potential pathogenic bacteria that act as endosymbionts.

This study was financially supported by Walailak University (Contract No 23/2562). The authors would like to also offer their thanks to Prof. Dr. Patrick Scheid and Dr. Carsten Balczun of Bundeswehr Central Hospital in Koblenz, Germany for their generosity in providing the positive controls used in this study; the School of Allied Health Sciences, Biomedical Science Department, Walailak University; Department of Biology and Department of Medical Technology, Far Eastern University for the technical assistance; laboratory technicians Mr. Joseph Dionisio, Romel Solomon, Virgilio Bitagcul, Ma. Lourdes Policarpio and Ivy Joy Fababier for providing technical support and Ms. Junee Linsangan of the UST Analytical Services Laboratory for providing technical support in electron microscopy. The authors declare that there is no conflict of interest.