Assessment of in vitro dynamics of pathogenic environmental Acanthamoeba T4 and T9 genotypes isolated from three recreational lakes in Klang Valley, Malaysia over the HaCaT cell monolayer

Acanthamoeba is a genus of aerobic protozoa thriving in numerous natural and artificial settings, such as fresh and tap water, soil, sand, dust, and sewage. The organism was also detected in healthy human nasal passages (Siddiqui & Khan 2012; Tawfeek et al. 2016). Acanthamoeba could survive as free-living protozoa or pathogens of vertebrates and hence are amphizoics (Fanselow et al. 2021). The species is among the most commonly isolated eukaryotic from the environment following the ability of its trophozoites to encyst, hence withstanding adverse surroundings (Alfieri et al. 2000). The Acanthamoeba have become relevant for public health, as they are natural hosts for numerous intracellular pathogens (Khan 2006). Furthermore, several species are correlated to two types of human illnesses: granulomatous amoebic encephalitis (GAE) and amoebic keratitis (AK) (Kot et al. 2018). The GAE is an opportunistic and often fatal infection affecting immunocompromised hosts. Conversely, AK affects healthy individuals, specifically contact lens wearers and might result in severe corneal damage (Cope et al. 2020).

Several Acanthamoeba species are classified according to their morphology and susceptibility to temperature (Pussard & Pons 1977). Nevertheless, not all the attributes are linked to human pathological occurrences. Although pathogenic potential determining factors are yet to be established entirely, several species such as Acanthamoeba culbertsoni, Acanthamoeba castellanii, Acanthamoeba polyphaga, Acanthamoeba hatchetti, and Acanthamoeba healy are more commonly detected in human infections (Schuster & Visvesvara 2004). Researchers internationally are employing a rapid and reliable detection approach based on the nuclear 18S small subunit ribosomal RNA gene to detect Acanthamoeba specimens (Maciver et al. 2013). Three remarkably informative Rns segments designated diagnostic fragments 1, 2, and 3 (DF1, DF2, and DF3) could yield robust phylogenetic trees, as they are based on the entire gene. Furthermore, the single significant variable and informative region, DF3 could allow prompt genotypic establishment (Kong 2009). As a typing criterion, strains under 5% difference in the 18S rRNA gene region are categorized as a single genotype (Siddiqui & Khan 2012). In total, 23 genotypes (T1–T23) have been identified via molecular characterization and phylogenetic analyses (Putaporntip et al. 2021). Some of the characterized genotypes are pathogenic, including T2, T3, T4, T5, T6, T11, and T15 with T4 as the most prevalent genotype isolated from human and environmental samples (Kalra et al. 2020). Identifying the genotypes of Acanthamoeba isolates from various ecological settings is essential for evaluating their virulence and tracking their epidemiological patterns, which can contribute to more effective public health measures.

Physiological and biochemical attributes and cytopathic effect (CPE) assays have allowed pathogenic and non-pathogenic Acanthamoeba differentiation (Mohd Hussain et al. 2022). Identifying pathogenic and non-pathogenic Acanthamoeba is critical for clinical diagnosis as the pathogenesis is correlated to numerous parameters. For instance, proteases are associated with host cell and tissue invasions and damage (Khan 2006). Pathogenic Acanthamoeba strains have higher temperature tolerance, growth rates and adherence characteristics, including greater cytotoxic product secretions and superior immune evasion mechanisms, than their non-pathogenic counterparts (Marciano-Cabral & Cabral 2003). Although Acanthamoeba isolates predominantly produce serine proteases, they also secrete distinctly patterned cysteine and metalloproteases (Khan 2006). Nonetheless, clinical isolates produce more extracellular proteases than environmental samples (Kim et al. 2006; Lorenzo-Morales et al. 2015). Several studies reported identifying 107 and 133 kDa serine proteases, significant virulence factors in pathogenic Acanthamoeba (Khan et al. 2000; Huang et al. 2017). Acanthamoeba isolates with significant pathogenic attributes also reflect a temperature tolerance of over 42 °C and exhibit remarkable proteolytic enzyme activities (Kot et al. 2018).

The trophozoites of different Acanthamoeba species spontaneously release proteinases into culture media, such as a plasminogen activator, collagenolytic enzymes, cysteine proteinases and possibly elastase-like enzymes and metalloproteinases (Carvalho-Silva et al. 2021). The enzymes result from contact-independent mechanisms, which might also assist parasitic human tissue colonization's. Regulating Acanthamoeba in the surroundings is crucial following its opportunistic nature and possible role as a human pathogen reservoir. The present study aimed to characterize the inherent pathogenic attributes of the Acanthamoeba samples. Physiological (thermo- and osmotolerance) and biochemical (proteolytic activities) evaluations were performed on the Acanthamoeba isolated from recreational lakes to verify their pathogenic potential. This study also determined the genotypes of the possibly pathogenic environmental isolates to associate pathogenicity with specific genotypes. This focus is crucial, as prior research has revealed significant differences in pathogenicity.

The current study employed 26 monoxenic Acanthamoeba isolates. In total, 10 samples were procured from Biru Lake (N: 3.2475°, E: 101.5263°) and eight samples each from Titiwangsa (N: 3.1781°, E: 101.7065°) and Shah Alam (N: 3.0729°, E: 101.5138°) Lakes. Each sample was inoculated centrally on a non-nutrient agar (NNA) plate (Sigma Aldrich, St. Louis, MO, USA), containing Page's amoeba saline (PAS) solution at pH 6.9. The agar was overlaid with ultraviolet (UV)-inactivated Escherichia coli (E. coli) (strain K12, ATCC, Manassas, VA, USA). The NNA plates were incubated at 30 °C and were microscopically observed daily with an inverted microscope under ×100 magnification for up to 72 h (Mohd Hussain et al. 2022). The present study also procured cloned cultures via dilution (Costa et al. 2010). A single trophozoite or cyst was employed for the cloning procedure. Subsequently, the cultures were transferred to a 1.5% NNA plate before conducting physiological assessments. This study employed A. castellanii (ATCC 50492) as the positive control.

The deoxyribonucleic acid (DNA) of the Acanthamoeba samples evaluated in the current study was extracted from its monoxenic culture with QIAamp^® DNA Mini Kit (Qiagen, Hilden, Germany). The methodology was applied following manufacturer guidelines. The DNA samples were stored at −20 °C until further use.

The polymerase chain reaction (PCR) assay genus-specific primer sets JDP1 and JDP2 employed in the present study were designed for Acanthamoeba genotyping (Schroeder et al. 2001). These primers, with sequences 5′-GGCCCAGATCGTTTACCGTGAA-3′ and 5′-TCTCACAAGCTGCTAGGGGAGTCA-3′ were established to amplify a fragment of 423–551 base pairs (bp) from the 18S rRNA region specific to Acanthamoeba, known as the Acanthameoba-specific (ASA.S1) amplimer. Each reaction was performed in triplicates with a final volume of 50 μL, consisting of 25 μL of TopTaq Master Mix (2×) (Qiagen, USA), 2 μL of each 10 μM of forward primers and 10 μM of reverse primers, 20 μL of DNase-free deionized water and 1 μL of DNA template (extracted DNA). The positive and negative controls in this study were prepared by incorporating the A. castellanii (ATCC 50492) DNA extract and DNase-free water (substituting the DNA template) into the reaction mix, respectively.

This study employed the GenJET PCR Purification Kit (Thermo Fisher Scientific, Waltham, MA, USA) to purify the PCR products based on the protocol outlined by the manufacturer. Subsequently, a BigDye^® Terminator v.3.1 Cycle Sequencing Kit (Thermo Fisher Scientific, USA) was utilized during forward and reverse sequencing. The resulting partial gene sequences of the 18S rRNA were subsequently analyzed using the Basic Local Alignment Search Tool (BLAST) program, hosted by the US National Center for Biotechnology Information (NCBI) website (https://www.ncbi.nih.gov/BLAST) to categorize the Acanthamoeba isolates into distinct species. This analysis involved comparing the obtained sequence with all confirmed Acanthamoeba genotypes in the GenBank database, accessed through the 80 NCBI platform (https://www.ncbi.nlm.nih.gov/pubmed) to determine the closest matching sequence (Mohd Hussain et al. 2022). Multiple sequence alignment of all Acanthamoeba-positive sequences obtained from this study, along with relevant reference sequences from GenBank was performed using ClustalW. These aligned sequences were then subjected to phylogenetic analysis using the MEGA software tool, v.11.0.13 (Mega Software, Tempe, Arizona, USA) (Tamura et al. 2013) with Balamuthia mandrillaris (NCBI KU184269) utilized as the outgroup. Phylogenetic trees were created based on the neighbour-joining distance tree method, which generated 1,000 bootstrapped replicates. The highest similarity percentages were recorded to establish the genotype and species.

In this study, thermo- and osmotolerance physiological evaluations were performed in triplicate. The cultures cultivated on an NNA medium enriched with E. coli were employed in the assessment following the methodology reported by Khan et al. (2001). Two culture plate sets were prepared for the assays.

In the thermotolerance evaluation, a small NNA block soaked with Acanthamoeba trophozoites or cysts was placed centrally on each culture plate. Subsequently, freshly prepared 1.5% NNA was overlaid with E. coli suspension before incubating the cultures at 37 and 42 °C. Small agar blocks consisting of Acanthamoeba cysts were sliced and positioned in the center of a fresh 1.5% NNA medium of 0.5 or 1 M of mannitol for osmotolerance assessment. The cultures were also overlaid with E. coli. The mannitol-free NNA plates were the negative control in this study.

The cultures were observed daily under a bright-field microscope at ×400 magnification over a period of 5 days. The proliferation of Acanthamoeba was assessed by counting all cysts or trophozoites located approximately 20 mm from the center of each plate. The mean counts of each sample were recorded from triplicate measurements. Growth was quantified based on the following categories: 0 (−), 1–15 (+), 16–30 (++ ), and >30 (+++). At the end of the analysis, samples were classified as high (+++), low (+ to ++ ) or non-pathogenic (−) based on previously published criteria (Landell et al. 2013). A. castellanii (ATCC 50492) was used as the reference strain to confirm pathogenic characteristics.

The trophozoites in this study were monitored daily for growth and collected after 3 days being subcultured on the NNA plates. The trophozoites were delicately procured from the agar surface of a minimum of three plates from each sample by adding 1–2 mL of sterile PAS solution. Gentamycin (100 g/mL) was also added upon washing the suspension twice with a cold PAS solution. Subsequently, the mixture was centrifuged at 3,500 rpm for 10 min.

The pellets obtained were lysed at 1.5 × 10⁷–2.5 × 10⁷ trophozoites/mL. The 0.15–0.20% (v/v) Triton^® X-100 prepared in water with or without proteinase inhibitor was incorporated, followed by one or two cycles of rapid freezing and thawing. The protein contents of the pellets were determined based on the procedure reported by Tawfeek et al. (2016), with slight modifications. Protein standards were prepared using bovine serum albumin (BSA) as the reference. The Bradford reagent was then added to both the prepared standards and Acanthamoeba lysate samples. These mixtures were incubated for 5–10 min at room temperature to allow for dye-protein interaction. After incubation, the absorbance was measured at 595 nm using a spectrophotometer. Finally, the protein concentration of each sample was determined by comparing the absorbance values against a standard curve generated from the BSA standards.

The current study utilized zymographic assays to determine the extracellular proteolytic reactions of the Acanthamoeba samples (Alfieri et al. 2000). Acanthamoeba trophozoite lysates were employed for the assessment. The zymography was prepared on 10% SDS-polyacrylamide gels copolymerized with 1% gelatin. The prepared sample buffer was mixed with the Acanthamoeba trophozoite lysate to yield a final volume of 30–40 μL (equivalent to 30 μg protein) before application on the gels.

A protein ladder of 10–250 kDa was used as the molecular size marker in the electrophoresis. Post-electrophoresis, the gels were soaked in 2.5% Triton^® X-100 (w/v) solution for 60 min and incubated at 37 °C in a developing buffer (50 mM of Tris-HCl, pH 7.5, 10 mM of CaCl₂) overnight. Finally, the gels were rinsed and stained with Coomassie Brilliant Blue R-250 (Bio-Rad Laboratories, Hercules, CA, USA). The electrophoresis gels were de-stained until clear, distinct, non-stained regions were observed against the dark blue background, indicating protease activities (Tawfeek et al. 2016). The de-staining process was terminated by applying a storage solution to the gels.

Protein molecular weights were determined using a gel documenting system software (Gel Analyzer 19.1). Similar experimental techniques were replicated on the trophozoite lysates from all samples. The isolate samples were pretreated with three protease inhibitors for 30 min before electrophoresis. In this study, 1 mM of phenylmethylsulphonyl fluoride (PMSF), 20 mM of N-ethylmaleimide (NEM), and 10 mM of ethylenediaminetetraacetic acid (EDTA) were employed as serine proteases, cysteine proteases, and metalloproteases inhibitors, respectively (Omana-Molina et al. 2013).

The present study procured human keratinocyte (HaCaT) cells (CLS Cell Lines Service, 300493) from the Cell Line Service GmbH (Eppelheim, Germany). The cells were grown in Dulbecco's Modified Eagle Medium (DMEM) (^©Capricorn Scientific, Ebsdorfergrund, Germany), which consisted of penicillin (100 U/mL), streptomycin (100 pg/mL) (Hi-Media Laboratories Pvt. Ltd, Maharashtra, India), and 10% fetal bovine serum (^©Capricorn Scientific, Ebsdorfergrund, Germany) (Mohd Hussain et al. 2022).

The HaCaT cells were preserved in a 5% carbon dioxide (CO₂) in air incubator at 37 °C and 80% humidity. After removing the existing media, 2 mL of trypsin was added to the confluent flasks (^©Capricorn Scientific, Ebsdorfergrund, Germany) to detach the cells. The cells were resuspended in new media and incubated in 24-well plates (NEST^®, Woodbridge, VA, USA). The samples were utilized in the cytotoxicity and cytopathogenicity assays after a homogenous monolayer was developed and verified microscopically within 48 h.

The HaCaT cell lines (CLS Cell Lines Service, 300493) cultured in this study were employed to determine the in vitro cytopathic effects (CPEs) of the Acanthamoeba samples (Mohd Hussain et al. 2022). The cells were grown in DMEM supplemented with penicillin (100 u/mL), streptomycin (100 pg/mL) (HiMedia Laboratories Pvt. Ltd, Maharashtra, India), and 10% fetal bovine serum (^©Capricorn Scientific, Germany) at 37 °C and 5% CO₂ in air for 24 h. The current study experiments were in quadruplicates, with 5 × 10⁵Acanthamoeba trophozoites employed per well on 24-well plates (NEST^®, USA). Each well contained a confluent cell monolayer with an amoebae-to-cell ratio of 1:2. The wells containing HaCaT cells that remained detached from the trophozoites were employed as control samples.

Crystal violet was utilized to stain the wells after being incubated for 24 h before performing a macroscopical assessment of visible alterations. In this study, the in vitro CPEs was analyzed based on the degree of monolayer cell damage by utilizing the ImageJ software. The results were categorized as no CPE (−), CPEs with up to 10% monolayer destruction (+), CPEs between 10 and 50% monolayer damage (++ ) and CPEs with 50–100% monolayer disruption (+++) (Possamai et al. 2018). The A. castellanii (ATCC 50492) strain was the positive CPE control in this study.

The HaCaT cells treated with 2% Triton^® X-100 for 1 h at 37 °C were employed to evaluate the total LDH released. Absorbance of the reduced salt, formazan (dye) was measured at 490 nm. A cytotoxicity of <10% was deemed non-toxic, while cytotoxicity between 10 and 25% was considered low toxic. Cytotoxicity between 25 and 40% was labeled intermediate, while levels over 40% were regarded as highly toxic (Lorenzo-Morales et al. 2010).

The ASA.S1 segment of the 18S rRNA gene molecular analysis indicated that the Acanthamoeba sampled from three recreational lakes were of the T4, T5, T9, T11, T17, and T18 genotypes (Table 1). The sequences obtained in the present study were submitted to GenBank under the accession numbers OQ247939–OQ247964.

The physiological assay of the amoebae samples evaluated in the present study demonstrated that 84.62% (22/26) of the isolates had thermo- and osmotolerance at 37 °C and 0.5 M of mannitol. Six T4 (B4, K2, K8, K9, SA1, and SA10), one T9 (K1) and one T11 (SA5) isolate were significantly pathogenic. The samples exhibited survivability at high temperatures (42 °C) and 1 M of mannitol osmolarity. Although the reference strain (A. castellanii ATCC 50492) utilized in this study survived the high temperature (42 °C) and osmolarity (1 M of mannitol), fewer cells were recorded than at 37 °C and 0.5 M of mannitol.

Two T4 (K8 and SA10) and one T9 (K1) isolate recorded 33, 42, and 133 kDa of enzyme proteases. Conversely, the B4 sample documented 56 and 133 kDa of protease enzymes (Figure 1). The addition of PMSF (serine protease inhibitor) hindered trophozoite protease activities entirely. Nevertheless, incorporating NEM (cysteine protease inhibitor) and EDTA (metalloprotease inhibitor) did not result in inhibitory effects against protease enzymes secreted by the Acanthamoeba trophozoites lysate from all isolates. The findings validated the different secretion patterns of serine proteases in the samples.

After 24 h, only three T4 (B4, K8, and SA10) and one T9 (K1) isolate incubated with the HaCaT cell monolayer documented cell destruction of over the 50–100% range (+++). The B4, K8, SA10, and K1 samples also recorded significant reactions toward physical stimuli. Furthermore, well plates consisting of over 50% trophozoite lysates from possibly pathogenic Acanthamoeba samples exhibited holes. Observably, the extracellular proteases produced by the Acanthamoeba isolates facilitated epithelial-cell disaggregation.

In total, 19 isolates assessed in the present study, including K2, K9, SA1, and SA5, exhibited thermo- and osmotolerance resistance. The samples demonstrated 10–50% (++ ) cell damage CPEs, while three isolates (K5, SA3, and SA4) recorded CPEs under 10% (+) (Table 3). Conversely, the HaCaT cell monolayer incubated with A. castellanii (ATCC 50492), the positive control sample documented 77.9% disruption. The monolayer without the amoebae, the negative control was the only sample without alterations.

The LDH results demonstrated that the potentially pathogenic Acanthamoeba isolates exhibited substantial in vitro cytopathogenicity levels in the HaCaT cell monolayer after 24 h of incubation (Table 3). Three T4 (B4, K8, and SA10) and one T9 (K1) genotype were highly toxic. The samples released over 40% LDH. Eight Acanthamoeba isolates, B5, B8, K2, K3, K7, K9, SA6, and SA7, recorded approximately 26–40% LDH releases, indicating intermediate cytotoxicity. Another 11 samples, B1, B2, B3, B6, B7, B9, B10, K6, SA1, SA2, and SA5 exhibited LDH releases between 11 and 25%, hence low cytotoxicity. Only three samples, K5, SA3, and SA4 released <10% LDH, which was non-toxic.

The ubiquity of the cosmopolitan Acanthamoeba protozoan could harm human health due to its capability to infect a host and survive in the environment. Acanthamoeba infections are associated with significant mortality and morbidity. Consequently, establishing the pathogen and initiating appropriate treatments immediately is critical. Nevertheless, data on the biological and cytopathogenic properties of different Acanthamoeba genotypes and their correlations with the virulence of each strain are limited.

The identification of Acanthamoeba genotypes T4, T5, T9, T11, T17, and T18 from three recreational lakes in Malaysia reflects both expected and novel ecological findings. The T4 genotype, which was frequently detected aligns with previous reports of its prevalence in diverse environments such as lakes and swimming pools and its strong association with Acanthamoeba keratitis infections, particularly among contact lens users (Siddiqui & Khan 2012; Rivera & Adao 2009). Genotypes T5, T9, and T11 although less commonly reported have also been isolated from aquatic environments, suggesting a diverse ecological community with varying pathogenic potentials (Booton et al. 2009). Notably, the presence of the rarer T17 and T18 genotypes may indicate site-specific environmental conditions or ecological niches that are underexplored, underscoring the need for further research into their distribution and pathogenic relevance (Lorenzo-Morales et al. 2015).

Eight T4 and singular T9 and T11 genotypes of Acanthamoeba isolates in this study were thermo- and osmotolerant, which are derivative virulence parameters. The findings paralleled the seven significantly thermo- and osmotolerant Acanthamoeba T4 isolates sampled from hot springs and beach water in Malaysia (Mohd Hussain et al. 2022). Predominantly, the T4 genotype obtained from Acanthamoeba in the environment demonstrated considerable thermo- and osmotolerance (Castro-Artavia et al. 2017). AK is primarily caused by genotype T4 (Satitpitakul et al. 2021). According to the defined sequence characteristics, T4 encompasses the T4A, T4B, T4C, T4D, T4E, T4F, and T4Neff subgroups (Fuerst & Booton 2020).

The T9 and T11 genotypes assessed in the present study were more capable of growing under harsh environments than the sample reported by Mohd Hussain et al. (2019). Furthermore, Acanthamoeba T11 isolate also demonstrated enhanced ability to grow in both extreme conditions, as reported by a number of previous studies (Todd et al. 2015; Milanez et al. 2020; Mohd Hussain et al. 2022). Additionally, a study carried out in Brazil discovered that Acanthamoeba T9 isolate was capable of flourishing in hyperosmotic and high-temperature environments, despite the fact that they were previously classified as non-pathogenic strains (Magliano et al. 2012). Nevertheless, it was important to highlight that, in studies conducted in Iran and India, T9 and T11 genotypes typically classified as non-pathogenic amoebae were the causal agents of AK and GAE (Hajialilo et al. 2016; Megha et al. 2018). This finding proved the pathogenic nature of the T9 genotype. Moreover, the T11 genotype which was scarcely reported worldwide was also identified in Chilean patients with AK (Jercic et al. 2019).

To be classified as potentially pathogenic, Acanthamoeba must exhibit thermo- and osmotolerant traits that reflect its behavior under stressful conditions (Todd et al. 2015). Their capacity to induce cellular damage in vitro is significantly correlated with growth at temperatures exceeding 40 °C (Walochnik et al. 2000), while proliferation under high mannitol concentrations indicates resistance to substantial osmotic pressure, which is critical in the corneal epithelium (Siddiqui & Khan 2012). However, isolates from similar genotypes may display varying pathogenic potential due to adaptability to environmental conditions, influenced by the release of heat shock proteins (HSP60 and HSP70) under stress (Solgi et al. 2012). Importantly, the presence of these traits does not define pathogenicity. For example, Possamai et al. (2018) found that certain thermotolerant strains were non-pathogenic. Kahraman & Polat (2024) reported that three of four T4B and T4E genotypes isolated from keratitis cases could not thrive at 39–41 °C and 1 M of mannitol, indicating that such tolerances do not equate to virulence. Given that the human cornea maintains a temperature of 32–35 °C, the pathogenicity of amoebae cannot be accurately assessed based solely on physiological characteristics, as they may still colonize hosts despite failing to grow above 37 °C (Mohd Hussain et al. 2022). Therefore, comprehensive pathogenicity assessments using diverse assays, such as the CPEs are essential to elucidate the impact of each Acanthamoeba genotype on human infections (Mohd Hussain et al. 2022).

Although proteases are critical in Acanthamoeba biology and pathogenesis, information on the enzymes is still inadequate (Khan 2006). Only a few investigations on a limited number of isolates of varying species and genotypes have been reported on Acanthamoeba extracellular proteases. Accordingly, the present study evaluated the extracellular serine proteases produced by all environmental isolates.

The migration patterns obtained in this study demonstrated bands with molecular weights from 27 to 248 kDa. The results confirmed the primary proteolytic enzymes previously detected in Acanthamoeba (Cirelli et al. 2020). The findings were comparable to other Acanthamoeba samples, including clinical cases (Khan 2006; Castro-Artavia et al. 2017). Three Acanthamoeba T4 (B4, K8, and SA10) isolates assessed in this study had 133 kDa serine protease. The observations supported a report where 133 kDa serine protease were detected in clinical isolates (Huang et al. 2017).

Huang et al. (2017) demonstrated that 133 kDa serine protease protein resulted in cytotoxic effects on human and hamster corneal epithelial cells. A 133 kDa serine protease, MIP133 was also vital in the Acanthamoeba pathogenesis pathogenic cascade. The protease promotes the degradation of keratocytes and iris ciliary body, retinal pigment epithelial, corneal epithelial and corneal endothelial cells and apoptosis of macrophage-like cells (Lorenzo-Morales et al. 2015). The 33 kDa enzyme detected in this study is critical in corneal tissue pathogenicity and invasions. The protease or a similar enzyme was identified in A. castellanii, A. healyi and A. lugdunensis (de Obeso Fernandez del Valle et al. 2023). Kim et al. (2006) suggested that the 33 kDa serine protease secreted by the keratopathogenic A. lugdunensis is crucial to AK pathogenesis, including corneal tissue invasion, immune evasion and nutrient uptake.

Mitra et al. (1995) reported that the approximately 42 kDa serine protease in Acanthamoeba belongs to the T4 genotype. The protease could be predominantly actin, which commonly correspond to approximately 20% of the total protein in Acanthamoeba (Pumidonming et al. 2014). The 56 kDa proteolytic enzyme was also detected in this study (genotype T4). The enzyme was detected in a contact lens-wearing keratitis patient in Spain (Heredero-Bermejo et al. 2015).

The diverse serine proteases within a single Acanthamoeba genotype might be attributed to strain, virulence, culture condition or assay method variations. Dudley et al. (2008) proposed that serine proteases play a role in nutrition and encystment and excystment, as they are required for eventual facultative parasitism by an amoeba. Khan (2006) highlighted the direct functional role of serine proteases in Acanthamoeba infections, demonstrating that intrastromal injections of the organism-conditioned medium resulted in in vivo corneal lesions similar to those observed in AK patients with reactions inhibited by PMSF, a serine protease inhibitor. Mechanistically, serine proteases contribute to Acanthamoeba virulence by degrading extracellular matrix (ECM) components such as collagen and elastin, thereby facilitating tissue invasion and enhancing the amoeba's ability to penetrate host tissues. Their enzymatic activities, including collagenase and elastinolytic functions, target key structural elements in the ECM, allowing the amoeba to evade immune responses and establish infections (Siddiqui & Khan 2012). Moreover, the ability of serine proteases to degrade plasminogen further promotes ECM breakdown and inflammation, exacerbating tissue damage. This enzymatic activity is particularly critical in conditions like keratitis and GAE, especially in immunocompromised individuals (Wang et al. 2023).

This study utilized HaCaT keratinocyte skin cells, a frequently employed cytolytic activity model, to determine probable in vitro cytotoxicity and CPEs of the Acanthamoeba samples (Anwar et al. 2019, 2020). Cell viability and cytotoxicity assays assess in vitro modifications at the cellular and metabolic levels by detecting structural alterations, such as membrane integrity loss or physiological and biochemical reactions correlated to non-viable and viable cells (Riss et al. 2013). Consequently, the approaches are useful in evaluating the cytotoxic effects of the Acanthamoeba isolates on human cells.

In this study, HaCaT monolayer disruptions were observed with crystal violet staining. The cultures were evaluated 24 h after adding B4, K1, K8, and SA10 or the control strain, A. castellanii (ATCC 50492). The disaggregation of the monolayer by amoebae trophozoites affixed to the plate in the spaces previously occupied by cells or between joined cells was primarily documented. The identical isolates and control strains also demonstrated a cytotoxicity level corresponding to the degree of cellular destruction observed in the crystal violet assay during the LDH release assessment. The study results coincided with the report by Martin-Navarro et al. (2010). Acanthamoeba Neff reportedly had 60–70% cytotoxicity, while Acanthamoeba CLC-16 had 55–75%.

Mohd Hussain et al. (2022) employed HeLa cell lines to determine cytotoxicity percentages of Acanthamoeba T4 (CL5, CL54, and CL149) and Acanthamoeba T3 (SKA5-SK35). The study reported significantly lower cytotoxicity levels, 50.9–60.6% (T4) and 50.2% (T3), than the results in the current study (59.4–70.3%). The difference could be related to the type of cell employed in the interaction.

The results in the present study suggested that the environmental samples, B4, K8, SA10 (genotype T4) and K1 (genotype T9), demonstrated an in vitro CPE revealing the cell monolayer disruptive abilities of the amoebae. The CPE evaluations in this study were conducted after subjecting the cultures to 12 passages, which took a maximum of 6 weeks (data unavailable). Although the procedure did not require a long monoxenic maintenance period, attenuation in CPE properties could not be discarded. Nevertheless, the detection of CPE even at low levels indicated pathogenicity, which was taken into account during analysis.

This study highlights important public health risks associated with recreational water use by identifying specific Acanthamoeba genotypes such as genotype T4, which is linked to infections like AK. These findings support more targeted risk assessments, helping health authorities monitor high-risk areas and populations. The currents study also provide valuable data for public awareness campaigns to educate recreational water users, particularly contact lens wearers on preventive measures. Additionally, the results can inform guidelines for water monitoring programs and safety protocols at recreational facilities. Identifying these genotypes lays the foundation for future surveillance efforts, offering insights into how environmental changes may influence the distribution and pathogenicity of Acanthamoeba over time.

In this study, only three recreational lakes were sampled, which may limit the generalizability of the findings regarding Acanthamoeba genotypic diversity. While these lakes were selected to reflect diverse human activities and environmental conditions, a broader sampling across additional lakes and geographical regions would provide a more comprehensive understanding of genotypic variation. Future research should consider expanding the scope to include lakes with different ecological conditions, human usage patterns and geographical locations. Moreover, since the data for this study were collected over a 1-year period, seasonal fluctuations and long-term trends in Acanthamoeba populations were not captured. This limitation may affect the interpretation of genotype prevalence and pathogenicity, as environmental conditions and human activity levels vary across seasons. Future research should consider implementing longitudinal studies over multiple years to account for seasonal variability and provide a more comprehensive understanding of Acanthamoeba population dynamics and potential pathogenicity.

In summary, the current study findings demonstrated virulence factors, suggesting the pathogenic potential of three Acanthamoeba T4 (B4, K8, and SA10) and a T9 (K1) genotype isolated from three recreational lakes in Peninsular Malaysia. Although amoebic pathogenicity could arise from intrinsic characteristics, Acanthamoeba infections might also indicate correlations between amoebic features, including growth temperature, proteolytic activity, and in vitro cytotoxicity. The present study provides novel insights and suggests a considerable variation in the response of Acanthamoeba members of the same genotype to physiological and biochemical pathogenicity indicators making generalization difficult. It is important to evaluate whether such differences exist among the different subtypes/species within the same genotype, especially those under genotype T4, which is implicated in the majority of Acanthamoeba infection. The significant LDH released (59.4%) by the environmentally isolated T9 genotype strain in the present study highlighted its infection-causing potential. The observations demonstrated that re-evaluating the role of other non-pathogenic Acanthamoeba genotypes in producing AK and other non-keratitis infections is necessary. The information would also provide novel and reliable diagnosis approaches and therapeutic strategies.

This study was financially supported by the Fundamental Research Grant Scheme (FRGS/1/2022/SKK0/UITM/02/13), Ministry of Higher Education, Malaysia and Geran Penyelidikan Penyelidik Muda Berbakat (600-RMC/YTR/5/3 [003/2021]), Universiti Teknologi MARA, Malaysia.

T.S.A. and R.H.M.H. contributed to the study conception and design. T.S.A. and R.A.H. implemented the study. R.A.H. performed the experiment. R.A.H., H.H., S.A., and R.H.M.H. analyzed and interpreted the data. T.S.A., N.A.K., and R.S. revised the work critically for intellectual content and granted final approval for publishing. All authors have reviewed the manuscript and consent was given to publish.

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

The authors declare there is no conflict.