Efficacy of propolis extract and eye drop solutions to suppress encystation and excystation of Acanthamoeba triangularis WU19001-T4 genotype

Preparation of propolis extracts

Ten samples of propolis were collected from different geographic regions of Iran (Table S1) with the permission and verification of plants from agricultural organizations in respective locations. The ethanolic extract of the plant was prepared by soaking 370 g of dry powder with 1 L of absolute ethanol for 7 days at room temperature. Afterwards, materials were filtered through Whatman filter paper No. 1 (GE Healthcare, Buckinghamshire, UK), the solvent was removed with a rotary evaporator (G3 Heidolph, Burladingen, Germany). Dried extracts were preserved at 4 °C and re-suspended in dimethyl sulfoxide (DMSO) at a 100 mg/mL concentration before use.

Eye drop solutions

Four patented commercially available eye drops solutions (EDS-1, -2, -3, and -4) for topical use against eye diseases were selected for analysis in this study. Details of the eye drops compositions are shown in Table S2.

Culture of A. triangularis WU19001 belonging to T4 genotype

A. triangularis WU19001 belonging to the T4 genotype (MW647650.1) was originally isolated from a recreational reservoir at Walailak University, Thailand. Trophozoites were grown in 75 cm² tissue culture flasks in PYG medium (proteose peptone 0.75% (w/v), yeast extract 0.75% (w/v) and glucose 1.5% (w/v)) without shaking at 28 °C as described previously (Mitsuwan et al., 2020). For cysts, trophozoites were transferred from the PYG medium to the Neff’s encystment medium (NEM; 0.1 M KCl, 8 mM MgSO₄·7H₂O, 0.4 mM CaCl₂·2H₂O, 1 mM NaHCO₃, 20 mM ammediol) and were cultured in this medium for 7 days. To obtain only mature cysts, culture cells were collected and then centrifuged at 4,000 rpm for 5 min. Supernatant was discarded and re-suspended with sodium dodecyl sulfate (SDS) solution at a final concentration of 0.5% (w/v) for 20 min to solubilize trophozoites and immature cysts (Moon et al., 2014). Then, mature cysts were harvested and washed twice using phosphate-buffer saline (PBS). All experiments were performed in line with the biosafety guidelines for scientific research at Walailak University, Thailand (Ref. No. WU-IBC-66-020).

Screening for anti-Acanthamoeba activity

All extracts were screened for anti-Acanthamoeba activity against A. triangularis WU19001 at a final concentration of 1 mg/mL by a broth microdilution method (Sangkanu et al., 2021). The stock solution (100 mg/mL) of the extract was prepared in DMSO and diluted to a concentration of 2 mg/mL with PYG medium. Aliquots of 100 µL were transferred to each well in triplicate. To prepare the final 1 mg/mL concentration, 100 µL of trophozoites or cysts (2 × 10⁵ cells/mL) were added. Plates were incubated at 28 °C for 24 h.

The percentage of cell viability analysis was the use of 0.2% trypan blue dye exclusion staining and observed under inverted microscopy (Nikon, Tokyo, Japan). The viability of parasites was calculated as follows: % viability = (mean of the viable parasite/mend of the control) × 100.

Determination of minimal inhibitory concentration

The minimal inhibitory concentration (MIC) of the active extracts was determined against the A. triangularis by broth microdilution methods as described previously (Mitsuwan et al., 2020). Briefly, 50 μL of two-fold serial dilutions in PYG medium, of the different extracts were added to each well. After that, 50 μL of Acanthamoeba trophozoites and cyst (2 × 10⁵ cells/mL) were seeded in the 96-well microtiter plates. The range of tested concentrations was 0.008 to 1 mg/mL. EDS were tested at concentrations of 100% and 50%. Chlorhexidine (Sigma-Aldrich, St. Louis, MA, USA) was tested in range of 0.001–0.064 mg/mL. The lowest concentration of propolis extract or EDS that inhibited 90% of A. triangularis growth was recorded as the MIC.

Inhibition of encystation on A. triangularis

Under unfavorable condition, Acanthamoeba trophozoites entry to cyst form with metabolically inactive and dormant cysts. Drugs that target Acanthamoeba are not effective against these cysts. Therefore, the inhibition of encystation was required. Encystation was performed as previously described (Dudley Alsam & Khan, 2008), with modifications. Briefly, propolis extracts or EDS at different concentrations (1/2 MIC, 1/4 MIC, 1/8 MIC, 1/16 MIC) were diluted with Neff’s medium in a 96-well plate and then Acanthamoeba trophozoites (5 × 10⁵ cells/mL) were added. Plates were incubated at 28 °C for 72 h. The total of amoeba number was counted using a haemocytometer (Boeco, Hamburg, Germany), then trophozoites and immature cysts were lysed by adding sodium dodecyl sulfate (SDS; 0.5% final concentration) and incubated for 1 h. After adding SDS, the surviving cysts were counted once again. The percentage of Acanthamoeba encystation was calculated as follows: (total number of amoebae post-SDS treatment/total number of amoebae pre-SDS treatment) × 100. Acanthamoeba alone served as the negative control, whereas PMSF (10 mM final concentration) was utilized as the positive control.

Inhibition of excystation on A. triangularis

Excystation is the emergence process of trophozoites from cysts resulting in infection recurring. For inhibition of excystation, Acanthamoeba cysts (5 × 10⁵ cells/mL) were incubated with various concentrations of propolis extracts and EDS (1/2 MIC, 1/4 MIC, 1/8 MIC, 1/16 MIC, 1/32 MIC and 1/64 MIC) in PYG medium in 96-well plate at 28 °C for 72 h. The excystation was determined using a hemocytometer and an inverted microscope. All of amoebae were counted, and then SDS (0.5% final concentration) was added and incubated for 1 h to dissolve the immature cysts and trophozoites. The remaining cysts in the cultures were counted again. The percentage of Acanthamoeba excystation was calculated using the following formula: (total number of amoebae pre-treatment with SDS−total number of amoebae post-SDS treatment)/(total number of amoebae pre-SDS treatment) × 100. PMSF (10 mM final concentration) and amoebae alone were used as positive and negative controls, respectively (Anwar et al., 2019).

Effects of combinations on inhibition of encystation and excystation

The propolis extract, alone or in combination with EDS was tested for anti-encystation and excystation. The propolis extract and EDS were diluted with Neff’s or PYG medium to obtain four times to their final concentrations of MIC, 1/2 MIC, 1/4 MIC, 1/8 MIC and 1/16 MIC in 96-well polystyrene plates (Sangkanu et al., 2021). Then, a total of 100 µL of Acanthamoeba trophozoites or cysts (5 × 10⁵ cells/mL) was added to each well. Plates were incubated at 28 °C for 72 h. Percentage detections of Acanthamoeba encystation and excystation were assessed using previous methods.

Effects of combination on anti-amoebic activity

The most active combination sets of propolis extract and EDS were integrated with chlorhexidine to determine the anti-amoebic activity. A total of 50 µL chlorhexidine were diluted in 96-well polystyrene plate (four times to their final concentrations of MIC, 1/2 MIC, 1/4 MIC, 1/8 MIC and 1/16 MIC). Then 50 µL of four times to final concentration of combination set was added. A total of 100 µL of trophozoites or cysts (2 × 10⁵ cells/mL) was added (Sangkanu et al., 2021). The plates were incubated for 24 h at 28 °C without agitation. Cell viability analysis involved dye exclusion staining followed by direct cell counting under the inverted microscopy (Nikon, Tokyo, Japan). Percentage amoebae viability was calculated from (mean of the treated parasite/mean of the control) × 100.

Calcofluor white staining

The cyst walls of Acanthamoeba can be stained blue white in this technique. Then, the results demonstrate that the calcofluor white stain method is a highly reliable technique for identification of Acanthamoeba cysts. Then, this technique was used for detection of Acanthamoeba cyst after treatment with propolis extract, EDS and combination solution. Acanthamoeba cells were incubated under inhibition of encystation condition (propolis extract No. 10, 0.016 mg/mL, and EDS-3, 6.25%) and excystation condition (propolis extract No. 10, 0.512 mg/mL, and EDS-3, 50%) for 72 h. After incubation, Acanthamoeba pellets were washed with PBS, re-suspended in a 2.5% calcofluor white staining solution, and incubated for 20 min at room temperature. After washing with PBS, samples were observed under a fluorescence microscope (BX53; Olympus, Tokyo, Japan) (Moon et al., 2013).

Scanning electron microscopy and transmission electron microscopy study

In this study, the morphology and surface of Acanthamoeba cells after treatment with combination of propolis extract and EDS was observed by SEM. Combination set of encystation included propolis extract No. 10 (0.016 mg/mL) and EDS-3 (6.25%) and combination set of excystation included propolis extract No. 10 (0.512 mg/mL) and EDS-3 (50%). After incubation for 72 h, parasites were collected by centrifugation at 3,000 rpm for 5 min and re-suspended in PBS. Samples were fixed with 2.5% glutaraldehyde for 2 h and then dehydrated with a series of graded ethanol (20%, 40%, 60%, 80%, 90%, and 100%). Dehydrated samples were mounted on aluminium stubs and allowed to dry using a critical point dryer EMS/K850 (Quorum, Laughton, UK). Samples were then coated with 99% gold particles with 108 Auto Sputter Coater (Ted Pella Inc., Redding, CA, USA) and the morphology of A. triangularis trophozoites and cysts in inhibition of encystation or excystation was subsequently examined under SEM (SEM-Zeiss, Munich, Germany) at the Center for Scientific and Technological Equipment, Walailak University, Nakhon Si Thammarat, Thailand.

Ultrastructure of Acanthamoeba after treatment with combined propolis extract and EDS was determined by TEM. Combination set of encystation included propolis extract No. 10 (0.016 mg/mL) and EDS-3 (6.25%) and combination set of excystation included propolis extract No. 10 (0.512 mg/mL) and EDS-3 (50%). Briefly, Acanthamoeba cells were harvested by centrifugation at 3,000 rpm for 5 min. The supernatants were discarded and the pellets were washed three times with PBS. The cell pellet was fixed in suspension in 2.5% glutaraldehyde. Post fixed in 1% osmium tetroxide and 2% uranyl acetate, then dehydrated with increasing concentrations of ethanol, and embedded in epoxy resin. After toluidine-blue stained sections, ultra-thin sections of 90 nm thickness were obtained from a diamond knife (Diatome) from the cell blocks, and the sections were stained with a 4% uranyl acetate-lead citrate solution. Cells were examined using the JEM 2010 transmission electron microscope (Jeol, Tokyo, Japan) at an accelerating voltage of 80 kV.

Cytotoxicity assay

The cytotoxic effects of the most active propolis extract, EDS, and combination sets were evaluated using the Vero cell line (8200F270602; Elabscience, Wuhan, Hubei, China). Vero cells are derived from the kidneys of African green monkeys and are widely used in research because of their adaptability and accessibility (Jethva & Bhatt Dhara Zaveri, 2016). The cells were grown in Dulbecco’s Modified Eagle’s (DMEM) medium (Merck KGaA, Darmstadt, Germany), which was enhanced with 10% FBS and 1% antibiotic supplemented with 100 units/mL of penicillin G and 100 μg/mL of streptomycin. The culture was incubated with 5% CO₂ at 37 °C in a humidified environment. At 90% confluence, cells were detached using trypsin and ethylene diamine tetra-acetic acid (EDTA).

A 96-well polystyrene plate was seeded with single cells at a density of 1.5 × 10⁴ cells/100 μL, and they were left to attach for 24 h. Next, a gentle addition of 100 μL of propolis extract, EDS, and combined set was added. After incubation for 24 h, the cytotoxic effects were determined using the MTT assay (Wilson et al., 2017; Mitsuwan, Wintachai & Voravuthikunchai, 2020). The absorbance was measured using a microplate reader (Biotek, Cork, Ireland) at 570 nm. The survival percentage was calculated using the following equation:

$\mathrm{\%}\mathrm{s}\mathrm{u}\mathrm{r}\mathrm{v}\mathrm{i}\mathrm{v}\mathrm{a}\mathrm{l}=(\mathrm{A}\mathrm{B}\mathrm{t}/\mathrm{A}\mathrm{B}\mathrm{u})\times 100.$

Cell survival was measured as the percentage absorbance compared with the treated cells (ABt) and non-treated cells (ABu).

Data analysis

Triplicate of the experiments were carried out. All information was gathered and entered into the statistical package (SPSS Inc., Chicago, IL, USA) software. The data was reported as mean ± SD. To perform statistical analysis, the two-tailed unpaired Student’s t-test was employed. In every analysis, a value of p < 0.05 was accepted as statistically significant.

Anti-Acanthamoeba activities

The anti-amoebic activities of propolis extracts on A. triangularis trophozoites and cysts were evaluated by calculating the percentage of viability. The activities observed at 24 h were compared to the untreated control, and the standard deviations (SD) are shown in Table S3. The percentage of viability rate of trophozoites in propolis extract from No. 1 (Tabriz city), No. 3 (Pranshahr city), No. 5 (Sarab city) and No. 10 (Kermanshah city) was 0. Furthermore, the cyst viability rate was 5.12 ± 7.94% when treated with 1 mg/mL of propolis extract No. 10. Based on this result, four propolis extracts showed good activity on A. triangularis were chosen for MIC determination.

Determination of the minimum inhibitory concentration

The MIC of active propolis extracts and EDS are summarized in Table S4. The sensitivities of trophozoites were significantly higher than cysts. Propolis extracts No. 1, No. 3, No. 5 and No. 10 exhibited MIC concentration in the trophozoites at 0.128–0.256 mg/mL, whereas propolis extract No. 10 (Kermanshah) showed good anti-Acanthamoeba activity in cysts with MIC at 1 mg/mL. Four EDS were also evaluated to determine the MIC concentration against A. triangularis. EDS-1, -2 and -3 expressed anti-Acanthamoeba activity with MIC at 100%, which inhibited the growth of trophozoites. Moreover, only EDS-3 revealed growth inhibition effects against trophozoites and cysts with MIC at 100%. EDS-4 did not show anti-Acanthamoeba activity on trophozoites and cysts. In the positive control, chlorhexidine exhibited MIC values of 0.008 and 0.032 mg/mL for trophozoites and cysts, respectively. Therefore, propolis extract No. 10 and EDS-3 were chosen for further study.

Inhibition of encystation in A. triangularis

To assess the effect of propolis extract No. 10 and EDS3 on A. triangularis encystation, PMSF was used as a positive control in Neff’s medium. According to the data presented in Fig. 1A, the results revealed that the propolis extract No. 10-exhibited inhibition of A. triangularis encystation at all concentrations. At 0.016 mg/mL (1/16 MIC) of propolis extract decreased encystation to 13.58 ± 2.2%. Inhibition of encystation in A. triangularis seems to be sensitive to EDS-3 at all concentrations (Fig. 1B). Seventy-two hours after the induction of encystation, the population of mature cysts was mainly present in the untreated control. In the PMSF, propolis extract, and EDS-3 treatments, the trophozoites were detached from the well and started to round and some cells were inverted into mature cysts (Fig. S1).

Effect of combination on inhibition of encystation

The percentage of A. triangularis encystation was challenged in EDS-3 combination with propolis extract No. 10 at various concentrations. After 72 h, 85.9 ± 4.6% of trophozoites switched to cyst form in the untreated control. The lowest percentage of encystation was seen at 3.6 ± 1.6% and 6.5 ± 2.2% when exposed to PMSF and in EDS-3 (6.25%) combination with propolis extract No. 10 (0.016 mg/mL), respectively compared to the untreated control (Fig. 1C).

Inhibition of excystation in A. triangularis

The effect of propolis extract and EDS treatment on excystation was assessed in PYG medium. Most trophozoites (60.1 ± 2.0%) emerged from cysts when incubated with propolis extract No. 10 at 0.064 mg/mL (1/16 MIC) and the excystation rate decreased (12.2 ± 2.2%) after exposure to high concentrations of propolis extract at 0.512 mg/mL (1/2 MIC) (Fig. 2A). For EDS, the lowest level of excystation (16.6 ± 2.0%) was observed when treated with 50% (1/2 MIC) of EDS-3 (Fig. 2B). Mature cysts with both layers of the cyst wall were seen as a major cell type in PMSF, propolis extract, and EDS treatment, while trophozoites were observed in the untreated control (Fig. S1).

Effect of combination on anti-excystation

The combination sets of propolis extract No. 10 and EDS-3 decreased the excystation of A. triangularis. The lowest percentage (9.4 ± 4.2%) of excystation was observed after incubation with 0.512 mg/mL of propolis extract No. 10 and 50% of EDS-3 compared to the untreated control (Fig. 2C).

Effect of combination on anti-amoebic activity

The mean of the percentage of viable trophozoites and cysts is presented in Table 1. There were fewer amoebae in the culture that received propolis extract No. 10, EDS-3 and chlorhexidine than in those treated with chlorhexidine alone under all conditions.

Calcofluor white staining

Calcofluor white staining was performed to determine the effects of propolis extract No. 10, and EDS-3 on cellulose synthase of A. triangularis. As shown in Fig. 3, calcofluor staining was not observed in trophozoites. Interestingly, 72 h post-encystation or excystation, positive calcofluor (blue color) was observed on cyst surfaces.

SEM and TEM study

Scanning electron micrographs of the parasite at various stages of encystation after exposure to the combination set of propolis extract No. 10 and EDS-3 are shown in Fig. 4. Trophozoites showed abundant acanthopodia on the surface (Fig. 4A). In the pre-encystation stage, trophozoites were round, which retained the short acanthopodia (Fig. 4B). Mature cysts exhibited an ostiole on the surface with a wrinkled cell wall (Fig. 4C) but some cysts were destroyed by propolis extract and EDS. Cysts exposed to combination set showed the morphological changes (Fig. 4D). At 72 h, loss of extracellular material was observed, which progressed to loss of cellular membrane integrity. The cellular membrane and cytoplasmic damage were evident. Ultrastructural changes within the Acanthamoeba cysts were examined using TEM and also confirmed the progressive destruction of A. triangularis cysts treated with propolis extract and EDS. Mature cyst of A. triangularis were presented in Neff’s medium (Fig. 5A), and some treated cysts had smaller size than in the control. Most had rough and thick cyst walls. Inside the cyst wall, there were shrinkage of the cytoplasm with lipid droplets lining the border of the cell. The space between the inner endocyst wall and the cytoplasm was filled (Fig. 5B). Some cells showed significant impairment of morphology. Most cells lost their compactness (Figs. 5C and 5D), ectocyst was no wrinkles (Fig. 5D), and cell was shrunken (Fig. 5E). In vitro excystation of A. triangularis was induced with propolis extract and EDS. The different stages of excystation were observed. A mature cyst showed a typical morphology which included an ostiole on the surface and a wrinkled cell wall (Fig. 6A). At the pre-emergence stage, the surface topology was indistinguishable from the mature cyst, and its ostioles were clearly observed (Fig. 6B). The remarkable feature of the post-emergence stage is the presence of empty cysts wall and trophozoites in the culture (Figs. 6C and 6D). Ultrastructural changes within the excysting cysts were examined using TEM. Mature cysts of A. triangularis exhibited the typical shape. The ectocyst was distinctly separated from the endocyst by an inter-cystic space, and the endocyst was noticeably separated from the cytoplasmic membrane. Ostioles and operculum were also clearly observed (Fig. 7A). Early stage of excystation, small dense granules were in the region close to the ostiole and associated with the cytoplasmic side of the plasma membrane (Fig. 7B), a less compact operculum (Fig. 7C), and the empty cyst (Fig. 7D) presented in the late phase of excystation. In addition, the irregular features were observed in treated cysts with propolis extract and EDS. The plasma membrane of the excysting cysts have been severely damaged, no defined the nuclear structures, plasm membrane shrunk significantly away from the walls of the endocyst. And there are micellar aggregations inside the cyst suggesting complete plasma membrane destruction (Figs. 7E and 7F).

Toxicity

At low concentrations, ranging from 0.008–0.064 mg/mL for propolis extract No. 10, the number of live cells was constant over 24 h. However, it had cytotoxic concentration values at 0.128–1 mg/mL that survival of Vero cells lower than 80%. To reduce the toxicity, the combined sets of propolis and EDS-3 were further challenged to determine the survival of Vero cells. A total of 80% of surviving Vero cells were observed at combined concentrations after 24 h of treatment (Fig. S2).