Acanthamoeba Castellanii Trophozoites Research Articles

The amoebicidal activity of three substances derived from benzothiazole on Acanthamoeba castellanii cysts and trophozoites and its cytotoxic potentials

Acanthamoeba species are free-living amoebae isolated from many ecological areas such as swimming pools, dams, lakes, soil, and air filters. These amoebae are usually causing granulomatous amebic encephalitis and amebic keratitis in immunosuppressive individuals. In this study, the reproductive potential and morphological changes determined of Acanthamoeba castellanii trophozoite and cyst forms exposed to three different active substances derived from benzothiazole. Furthermore, the cytotoxic potential of these active substances determined by XTT analysis. In the study, axenic cultures prepared for Acanthamoeba castellanii cyst and trophozoite forms and parasite exposed to different concentrations of active substances. Cell counts of parasite cultures were performed at the 30 minutes, 1st, 6th, 12th, 24th, and 48th hour periods. As a result of the study, the reproductive potential suppressive effects of all three substances on Acanthamoeba castellanii trophozoites and cysts were determined. The most effective of these substances was 2-Amino-6(trifluoromethoxy)-benzothiazole. In the first three concentrations of this substance (0.1%, 0.05%, 0.025%), no determined trophozoite and cysts at the end of twenty four. Due to its strong ameobicidal effect, it is thought that 2-Amino-6(trifluoromethoxy)-benzothiazole may be a new therapeutic agent in diseases caused by acanthamoeba parasites by supporting this study with animal experiments.

Morphological Description of the Early Events during the Invasion of Acanthamoeba castellanii Trophozoites in a Murine Model of Skin Irradiated under UV-B Light.

Skin infections have been associated with Acanthamoeba, nevertheless the events during skin invasion and UV-B light effects on it are unknown. The early morphological events of Acanthamoeba castellanii skin invasion are shown in SKH-1 mice that were chronically UV-B light irradiated. Mice that developed skin lesions (group 1) were topical and intradermally inoculated with A. castellanii trophozoites and sacrificed 48 h or 18 days later. Mice that showed no skin lesions (group 2) were intradermally inoculated and sacrificed 24, 48 or 72 h later. Mice ventral areas were considered controls with and without trophozoites intradermally inoculated. Skin samples were processed by histological and immunohistochemistry techniques. In group 1, trophozoites were immunolocalized in dermal areas, hair cysts, sebaceous glands, and blood vessels, and collagen degradation was observed. One of these mice shown trophozoites in the spleen, liver, and brain. In group 2, few trophozoites nearby collagenolytic activity zones were observed. In control samples, nor histological damage and no trophozoites were observed. Adherence and collagenolytic activity by A. castellanii were corroborated in vitro. We can infer that UV-B light irradiated skin could favor A. castellanii invasiveness causing damage in sites as far away as the brain, confirming the invasive capacity and pathogenic potential of these amphizoic amoebae.

In vitro activity of Camellia sinensis (green tea) against trophozoites and cysts of Acanthamoeba castellanii

The effect of Camellia sinensis (green tea) on the growth of Acanthamoeba castellanii trophozoites was examined using a microplate based-Sulforhodamine B (SRB) assay. C. sinensis hot and cold brews at 75% and 100% concentrations significantly inhibited the growth of trophozoites. We also examined the structural alterations in C. sinensis-treated trophozoites using transmission electron microscopy (TEM) and scanning electron microscopy (SEM). This analysis showed that C. sinensis compromised the cell membrane integrity and caused progressive destruction of trophozoites. C. sinensis also significantly inhibited the parasite's ability to form cysts in a dose-dependent manner and reduced the rate of excystation from cysts to trophozoites. C. sinensis exhibited low cytotoxic effects on primary corneal stromal cells. However, cytotoxicity was more pronounced in SV40-immortalized corneal epithelial cells. Chromatographic analysis showed that both hot and cold C. sinensis brews contained the same number and type of chemical compounds. This work demonstrated that C. sinensis has anti-acanthamoebic activity against trophozoite and cystic forms of A. castellanii. Further studies are warranted to identify the exact substances in C. sinensis that have the most potent anti-acanthamoebic effect.

Isoniazid Conjugated Magnetic Nanoparticles Loaded with Amphotericin B as a Potent Antiamoebic Agent against Acanthamoeba castellanii.

The pathogenic free-living amoeba, Acanthamoeba castellanii, is responsible for a rare but deadly central nervous system infection, granulomatous amoebic encephalitis and a blinding eye disease called Acanthamoeba keratitis. Currently, a combination of biguanides, amidine, azoles and antibiotics are used to manage these infections; however, the host cell cytotoxicity of these drugs remains a challenge. Furthermore, Acanthamoeba species are capable of transforming to the cyst form to resist chemotherapy. Herein, we have developed a nano drug delivery system based on iron oxide nanoparticles conjugated with isoniazid, which were further loaded with amphotericin B (ISO-NPs-AMP) to cause potent antiamoebic effects against Acanthamoeba castellanii. The IC50 of isoniazid conjugated with magnetic nanoparticles and loaded with amphotericin B was found to be 45 μg/mL against Acanthamoeba castellanii trophozoites and 50 μg/mL against cysts. The results obtained in this study have promising implications in drug discovery as these nanomaterials exhibited high trophicidal and cysticidal effects, as well as limited cytotoxicity against rat and human cells.

Amoebicidal activity of benzothiazole on Acanthamoeba castellanii cysts and trophozoites and its cytotoxic potentials

Acanthamoeba spp. are free-living amoebae isolated from many ecological areas such as swimming pools, dams and lakes, and soil. Granulomatous amebic encephalitis and amebic keratitis, caused by Acanthamoeba spp., usually occurs in chronically ill, debilitated individuals, in immunosuppressed patients and treatment is quite difficult. This study aimed to determine the effect of benzothiazole on trophozoite and cyst forms of Acanthamoeba castellanii (A.castellanii). Axenic cultures of A. castellanii trophozoites and cysts were prepared to test the amoebicidal activity of benzothiazole. The concentrations of benzothiazole in 24-well plates were prepared as 0.08%, 0.04%, 0.02%, 0.01%, 0.005%, and A. castellanii cysts and trophozoites were added to these cultures. Parasites were counted at 0, ½, 1, 3, 6, 12, 24 and 48 h in the cell counter after staining with trypan blue. Cytotoxicity of benzothiazole on the WI-38 human fibroblast cell line was also tested. Between 0.08% and 0.01% concentrations of benzothiazole showed a strong amoebicidal activity at 24 and 48 h. A significant decrease in 0.005% concentration in the number of live trophozoites and cysts was detected between 6 and 48 h. As a result of the cytotoxicity studies, benzothiazole did not show any cytotoxic effect on the WI-38 human fibroblast cell line even at 1% concentration. Benzothiazole could be concluded as a new therapeutic agent against Acanthamoeba. On the other hand, in vivo studies are needed to confirm the efficacy of the biological effect.

Comparison of in vitro assays to study the effectiveness of antiparasitics against Acanthamoeba castellanii trophozoites and cysts

AbstractPurposeTo compare LDH release assay, trypan blue and fluorescent stainings and non‐nutrient Escherichia coli (E. coli) plate assay in determining treatment efficacy of antiamoebic‐agents against Acanthamoeba castellanii trophozoites and cysts, in‐vitro.MethodsA. castellanii strain 1BU‐trophozoites/cysts were challenged with the antiamoebic‐agents 0.02% polyhexamethylen‐biguanid (PHMB), 0.1% propamidin‐isethionate (PD) and 0.0065% miltefosine (MF). Efficacies of the drugs were determined by LDH‐release and trypan blue assays. The fluorescent dyes Hoechst 33343, calcein‐AM, and ethidium‐homodimer‐1 were tested for their abilities to differentiate between viable and dead 1BU‐trophozoites/cysts following treatment, a non‐nutrient agar E. coli plate assay was applied to monitor the outgrowth of 1BU‐trophozoites/cysts challenged with antiamoebic‐agents.ResultsAll three antiamoebic‐agents induced a significant LDH‐release from trophozoites, when compared to controls (p < 0.0001). There was a negligible trypan blue, Hoechst33342, calcein‐AM and ethidium‐homodimer‐1 staining in untreated 1BU‐throphozoites and cysts. Percentage of trypan blue positive trophozoites/cysts was 59.3 ± 1.5% /100% after challenge with PHMB, 100%/99.3 ± 0.5% upon PD treatment, and 95.6 ± 2.5%/77.6 ± 5.1% when using MF. Hoechst 33342 stained 100%/100%, ethidium‐homodimer‐1 100%/100%, calcein‐AM 75.33 ± 4.04%/60.67 ± 3.51% of PHMB treated trophozoites/cysts. Hoechst 33342 and ethidium‐homodimer‐1 were 100%/100%, calcein‐AM 9.67 ± 2.52%/36.67 ± 4.51% positive in PD‐treated trophozoites/cysts. MF treatment resulted in 100%/100% Hoechst 33342 and ethidium‐homodimer‐1 positive trophozoites/cysts, while none of them became positive for calcein‐AM. On non‐nutrient agar E. coli plates, in controls and MF treated 1BU trophozoites/cysts, new trophozoites appeared within 24 hr, and encystment occured after 5 weeks of cultivation. In PHMB and PD treated 1BU‐throphozoites/cysts, irregularly shaped, smaller trophozoites appeared after 72 hr, which failed to form new cysts within 5 weeks.ConclusionsNone of the enzymatic‐ and dye‐based viability assays tested here generated survival rates for trophozoites/cysts that were comparable with those yielded with the non‐nutrient agar E. coli plate assay, suggesting that the culture‐based assay is the best method to study the treatment efficacy of drugs against Acanthamoeba.

Cytopathic Change and Inflammatory Response of Human Corneal Epithelial Cells Induced by Acanthamoeba castellanii Trophozoites and Cysts

Acanthamoeba castellanii has ubiquitous distribution and causes primary acanthamoebic keratitis (AK). AK is a common disease in contact lens wearers and results in permanent visual impairment or blindness. In this study, we observed the cytopathic effect, in vitro cytotoxicity, and secretion pattern of cytokines in human corneal epithelial cells (HCECs) induced by A. castellanii trophozoites and/or cysts. Morphological observation revealed that panked dendritic HCECs co-cultured with amoeba cysts had changed into round shape and gradually died. Such changes were more severe in co-culture with cyst than those of co-cultivation with trophozoites. In vitro cytotoxicity assay revealed the highest cytotoxicity to HCECs in the co-culture system with amoeba cysts. A. castellanii induced the expression of IL-1α, IL-6, IL-8, and CXCL1 in HCECs. Secreted levels of IL-1α, IL-6, and IL-8 in HCECs co-cultured with both trophozoites and cysts were increased at an early incubation time (3 and 6 hr). These results suggested that cytopathic changes and pro-inflammatory cytokines release of HCECs in response to A. castellanii, especially amoebic cysts, are an important mechanism for AK development.

N-chlorotaurine Inactivates Acanthamoeba and Candida albicans in the Porcine Ex Vivo Corneal Infection Model.

N-chlorotaurine (NCT) is an anti-infective belonging to the class of chloramines and an investigative drug for the topical treatment of keratoconjunctivitis. The aim of the present study was to demonstrate its efficacy against Acanthamoeba and Candida in corneas infected ex vivo. Corneal buttons from porcine eyes were contaminated with Acanthamoeba castellanii trophozoites or Candida albicans Centraalbureau voor Schimmelcultures 5982 and incubated for 7 and 3 days, respectively. Subsequently, they were treated with 1% NCT for 5 to 120 minutes. After further incubation for 2 days in the absence of NCT in tests with A. castellanii, the buttons were homogenized, and the amoebae grown for a further 5 days before they were counted in a light microscope. For C. albicans, quantitative cultures were performed from corneal homogenates. Incubation of 120 minutes in NCT completely inhibited the regrowth of A. castellanii and reduced the number of C. albicans colony-forming unit counts by 4 log10. In addition, at 60 minutes, significant reductions of both pathogens could be observed. Histology showed penetration of pathogens into the stroma of the corneal buttons. NCT inactivates A. castellanii and C. albicans in corneal tissue.

3D Hydrogels Containing Interconnected Microchannels of Subcellular Size for Capturing Human Pathogenic Acanthamoeba Castellanii.

Porous hydrogel scaffolds are ideal candidates for mimicking cellular microenvironments, regarding both structural and mechanical aspects. We present a novel strategy to use uniquely designed ceramic networks as templates for generating hydrogels with a network of interconnected pores in the form of microchannels. The advantages of this new approach are the high and guaranteed interconnectivity of the microchannels, as well as the possibility to produce channels with diameters smaller than 7 μm. Neither of these assets can be ensured with other established techniques. Experiments using the polyacrylamide substrates produced with our approach have shown that the migration of human pathogenic Acanthamoeba castellanii trophozoites is manipulated by the microchannel structure in the hydrogels. The parasites can even be captured inside the microchannel network and removed from their incubation medium by the porous polyacrylamide, indicating the huge potential of our new technique for medical, pharmaceutical, and tissue engineering applications.

Comparative proteomic analysis of soluble and surface-enriched proteins from Acanthamoeba castellanii trophozoites

Acanthamoeba castellanii is a free-living organism widely distributed in the environment that may cause disease. This protozoan exists in two forms, an infective trophozoite and a dormant cyst. The trophozoites are able to cause keratitis and granulomatous amoebic encephalitis in humans. Keratitis is an acute, sight threatening infection of cornea with potential to cause permanent blindness without prompt treatment. However, the lack of suspicion and the low awareness about these amoebae besides of the absence of commercially available immunodiagnostic tests may delay an accurate diagnosis. The identification of proteins with potential for use in immunodiagnosis may improve the parasite detection more quickly and specifically. The amoeba adhesion to the host cell is the primary step for infection but there is no full understanding of A. castellanii proteins relevant for host invasion or infection. In this study, an assessment of soluble and surface-enriched protein fractions expressed by A. castellanii trophozoites, based on complementary LC–MS/MS approach using peptides from SDS-PAGE excised bands, was performed. Our proteomic analysis allowed identification of a total of 503 proteins, of which 308 proteins were exclusively identified in the soluble fraction, 119 in surface-enriched fraction and 76 in both. In silico analysis of functional classification revealed several proteins involved in many biological mechanisms in A. castellanii, including pathogen survival and infection of mammalian hosts. The analysis of predicted antigenic peptides allowed the identification of proteins with potential for immunodiagnostic assays.

Optical sectioning in multifoci Raman hyperspectral imaging

AbstractIn this study, we compared the depth discrimination and speed performance of multifoci Raman hyperspectral imaging with the reference standard of a single laser point confocal Raman mapping. A liquid crystal spatial light modulator was employed for the generation of multifoci laser beams, and a digital micromirror device was used as a software‐configurable reflective pinhole array. The patterns of the laser foci and pinhole array can be rapidly changed without requiring any hardware alterations. Confocal patterns with different distance‐to‐size ratios were tested and compared. After optimization of the laser‐foci pattern, we demonstrated the feasibility of multifoci Raman hyperspectral microscopy for recording depth‐resolved molecular maps of biological cells (Acanthamoeba castellanii trophozoites). Micrometric depth discrimination and short acquisition times (20 min for single plane confocal image) were achieved.

Investigation of in vitro amoebicidal activities of Ornithogalum sigmoideum on Acanthamoeba castellanii cysts and trophozoites

Aim: In this study, amoebicidal activity of methanol and ringer extracts obtained from Ornithogalum sigmoideum against Acanthamoeba castellanii cyst and trophozoite forms was investigated by determining cell viability percentage and IC50 values. Material and Methods: The in vitro amoebicidal activities methanolic (1%) and Ringer extracts of O. sigmoideum prepared at varying concentrations (in the range of 40 mg/ml -1.25 mg/ml) on A. castellaniitrophozoites and cysts were investigated. Results: IC50 results observed at 1st, 3rd, 6th, 8th, 24th, 48th and 72nd hours in A. castellaniitrophozite form were 23.2, 15.6, 12.1, 10.1, 8.3, 6.6 and 2.9 mg/ml by using the methanolic extract of O. sigmoideum, and 24.2, 16.7. 13.3, 11.9, 8.9, 8.4 and 7.3, mg/ml by using the Ringer extract ofsigmoideum respectively. The 40 mg/ml methanolic extract of O. sigmoideum killed all trophozoites at 48th and 72nd hours. The % viability rate at 72nd hour when using the Ringer extract of O. sigmoideum was 2.0±0.0. Conclusion: The methanolic extract was found to be more effective than the Ringer extract on Acanthamoebatrophozoites. The sensitivity of A. castellaniicysts to both extracts was not very different from each other, and the sensitivity of trophozoites to both extracts was higher than that of cysts. The methanolic extracts of O. sigmoideum can be used as an alternative treatment option or in combination with other therapies for treating Acanthamoeba infections. In addition, Ringer extract of O. sigmoideum can also be used as an alternative to the methanolic extract.

Apoptosis of Acanthamoeba castellanii Trophozoites Induced by Oleic Acid.

Acanthamoeba spp. can be parasitic in certainsituations and are responsible for serious human infections, including Acanthamoeba keratitis, granulomatous amoebic encephalitis, and cutaneous acanthamoebiasis. We analyzed the fatty acid composition of Acanthamoeba castellanii trophozoites and tested the inhibitory activity of the main fatty acids, oleic acid and arachidonic acid, invitro. Oleic acid markedly inhibited the growth of A.castellanii, with trophozoite viability of 57.4% at a concentration of 200μM. Caspase-3 staining and annexin V assays showed that apoptotic death occurred in A.castellanii trophozoites. Quantitative PCR and dot blot analysis showed increased levels of metacaspase and interleukin-1β converting enzyme, which is also an indication of apoptosis. In contrast, arachidonic acid showed negligible inhibition of growth of A.castellanii trophozoites. Stimulated expression of Atg3, Atg8 and LC3A/B genes and monodansylcadaverine labeling suggested that oleic acid induces apoptosis by triggering autophagy of trophozoites.

Effect of Multipurpose Solution Combined With Autophagy Inhibitors on Adhesion of Acanthamoeba trophozoites to Silicone Hydrogel Contact Lenses.

The aim of the study was to evaluate the effect of multipurpose contact lens (CL) solution (MPS) combined with autophagy inhibitors on the adhesion properties of Acanthamoeba castellanii (AC) trophozoites to silicone hydrogel CLs. AC trophozoites were inoculated onto discs trimmed from a silicone hydrogel CL with plasma surface treatment. After 18 hours, the number of adherent AC trophozoites on the CL treated with MPS was counted under phase-contrast microscopy. We analyzed the efficacy of MPS combined with autophagy inhibitors, 3-methyladenine (0.1, 1, and 5 mM) and chloroquine (10, 100 μM, and 1 mM), on Acanthamoeba adhesion by electron microscopy. Adhesion of AC trophozoites to the CL treated with MPS (average number of trophozoites adhered to the CL: 61.2 ± 8.1) was significantly lower compared with that of the CL treated without MPS (83.8 ± 10.2) (P = 0.027). In MPS application, the number of adhered AC trophozoites treated with 3-methyladenine [8.2 ± 2.5 for 5 mM (P = 0.008)] or chloroquine [19.4 ± 7.6 for 100 μM (P = 0.038) and 5.3 ± 1.9 for 1 mM (P = 0.001)] was significantly reduced compared with the sample without autophagy inhibitors. However, the number of adherent AC trophozoites was not significantly reduced in less than 5 mM in 3-methyladenine [50.4 ± 5.1 for 0.1 mM (P = 0.084) and 43.1 ± 5.0 for 1 mM (P = 0.079)] and 100 μM in chloroquine [40.6 ± 13.5 for 10 μM (P = 0.075)]. 3-methyladenine induced blebby structures or disrupted the membranes of AC trophozoites. AC trophozoites treated with chloroquine showed undigested organelles in the cytoplasm of Acanthamoeba cells. MPS combined with 3-methyladenine or chloroquine reduced the adhesion rate of AC trophozoites rather than MPS containing only polyhexamethylene biguanide. Appropriate concentrations of autophagy inhibitors, 3-methyladenine and chloroquine, added to commercial MPS should be considered to decrease the clinical rate of Acanthamoeba keratitis.

Efficacy of Korean Multipurpose Contact Lens Disinfecting Solutions against Acanthamoeba castellanii.

Acanthamoeba keratitis has been increasing in recent years. Main risk factors are contact lens wear and their cleaning solutions. Most contact lens wearers use multipurpose disinfecting solutions (MPDS) for cleansing and disinfecting microorganisms because of its convenience. We determined amoebicidal effects of MPDS made in Korea and their cytotoxicity on human corneal epithelium cells. Fifteen commercial MPDS (A to O) were tested for their amoebicidal effects on Acanthamoeba castellanii trophozoites and cysts by using a most probable number (MPN) technique. Among them, 7 kinds of MPDS showed little or no amoebicidal effects for 24 hr exposure. Solutions A, B, G, H, L, and O showed positive amoebicidal effects, and solutions M and N killed almost all trophozoites and cysts after 24 hr exposure. However, 50%-N solution showed 56% cytotoxicity on human corneal epithelial cells within 4 hr exposure, and 50%-O solution also showed 62% cytotoxicity on human cells within 4 hr exposure. Solution A did not show any cytotoxicity on human cells. These results revealed that most MPDS made in Korea were ineffective to kill Acanthamoeba. The solutions having amoebicidal activity also showed high levels of cytotoxicity on human corneal epithelial cells. New formulations for improved MPDS that are amoebicidal but safe for host cells are needed to prevent Acanthamoeba keratitis.

Investigation of the in vitro effects of Melissa officinalis L., Mentha x piperita L. and Ocimum basilicum L. (Lamiaceae) essential oils on the cysts and trophozoites of Acanthamoeba castellani

Acanthamoeba species are free living amoeba found widely all over the world. They are responsible for Acanthamoeba keratitis (AK), an infection which is especially seen in contact lens users and after minor corneal traumas, that may lead blindness. At present, antifungals and antiseptics are used for the treatment of AK cases, however, some problems such as long treatment periods and the occurrence of side effects, resistance of cyst forms against drugs, emphasize the need for new drugs. There are some published studies that pointed out the effectiveness of plant extracts and essential oils on Acanthamoeba spp. The aim of this study was to investigate the in vitro effects of essential oils of Mentha x piperita L. (peppermint), Melissa officinalis L. (lemon balm) and Ocimum basilicum L. (basil) belonging to Lamiaceae family, on the cysts and trophozoites of Acanthamoeba castellanii. The strain used in our study, namely A. castellanii T4 genotype, is the most frequently isolated amoeba from environment and also the causative agent of AK and granulomatous amebic encephalitis. For the determination of amebicidal activity, essential oils obtained from Mentha x priperita L., Melissa officinalis L. and Ocimum basilicum L. by Neo-Clevenger type of distillation apparatus have been used. In vitro experiments were performed by using 96-well microplates. Cyst and trophozoite solutions were added on the essential oil dilutions to obtain the last concentrations of 40, 20, 10, 5, 2.5 and 1.25 µg/ml for the cysts, and 10, 5, 2.5, 1.25, 0.625 and 0.313 µg/ml for the trophozoites. After the incubation of microplates at 30oC for 1, 6, 24, 48 and 72 hours, the viability of parasitic forms were evaluated under the light microscope followed by staining trypan blue. It was found that, each essential oil showed amebicidal effect on A.castellani cysts and trophozoites dependent on dosage and time, when compared with the control group, The maximum lethal effect occured with Melissa officinalis followed by Mentha x piperita and Ocimum basilicum, respectively. In our study, susceptibility of A.castellanii trophozoites to essential oils were more than the cysts, as expected. The essential oils of Melissa officinalis and Mentha x piperita showed 100% lethal effect at their highest concentrations whereas the essential oil of Ocimum basilicum showed only 63.3% lethal effect on cysts after 72 hours at the highest concentration (40 µg/mL). The results of this first study investigating the activities of essential oils extracted from Mentha x piperita, Melissa officinalis and Ocimum basilicum against Acanthamoeba spp. cysts and trophozoites, have suggested that, these essential oils could be potential novel and alternative natural products for the treatment of Acanthamoeba spp. infections.

Effects of multipurpose solutions on the adhesion of Acanthamoeba to rigid gas permeable contact lenses.

To evaluate the effect of multipurpose contact lens care solutions (MPSs) on the adhesion of Acanthamoeba to rigid gas permeable (RGP) contact lenses. Acanthamoeba castellanii (AC) trophozoites were inoculated onto untreated RGP contact lenses (FP, Extra, or Menicon Z), and numbers of trophozoites adhering to lenses were counted under a phase contrast microscope at 18h post-inoculation (controls). Similarly, adhering trophozoites were counted at 6h post-inoculation on each of the three RGP lens types with one of three MPSs (Boston Simplus, Menicare Plus, and O2 Care). Scanning electron microscopic examinations were performed to compare lens surfaces. Adhesion of AC trophozoites to untreated FP was greater than to untreated Extra or Menicon Z. Surfaces of Extra and Menicon Z lenses were waxier, smoother, and more homogeneous than those of FP lenses. After treatment with Boston Simplus, adhesion of AC trophozoites was significantly reduced for all lens types as compared with controls (p<0.0001). Treatments with Menicare Plus or O2 Care reduced the number of adherent AC trophozoites significantly on FP lenses only as compared with controls (p<0.0001). The adhesion rates of AC trophozoites to RGP lenses depended on lens surfaces. Boston Simplus reduced the adhesion rate of AC trophozoites more than Menicare Plus or O2 Care. Appropriate RGP lens and MPS selection could decrease the prevalence of Acanthamoeba keratitis.

Interactions of Pseudomonas aeruginosa and Corynebacterium spp. with non-phagocytic brain microvascular endothelial cells and phagocytic Acanthamoeba castellanii.

Several lines of evidence suggest that Acanthamoeba interact with bacteria, which may aid in pathogenic bacterial transmission to susceptible hosts, and these interactions may have influenced evolution of bacterial pathogenicity. In this study, we tested if Gram-negative Pseudomonas aeruginosa and Gram-positive Corynebacterium spp. can associate/invade and survive inside Acanthamoeba castellanii trophozoites and cysts, as well as non-phagocytic human brain microvascular endothelial cells. The results revealed that both Corynebacterium spp. and P. aeruginosa were able to associate as well as invade and/or taken up by the phagocytic A. castellanii trophozoite. In contrast, P. aeruginosa exhibited higher association as well as invasion of non-phagocytic HBMEC compared with Corynebacterium spp. Notably, P. aeruginosa remained viable during the encystment process and exhibited higher levels of recovery from mature cysts (74.54 bacteria per amoebae) compared with Corynebacterium spp. (2.69 bacteria per amoeba) (P < 0.05). As Acanthamoeba cysts can be airborne, these findings suggest that Acanthamoeba is a potential vector in the transmission of P. aeruginosa to susceptible hosts. When bacterial-ridden amoebae were exposed to favourable (nutrient-rich) conditions, A. castellanii emerged as vegetative trophozoites and remained viable, and likewise viable P. aeruginosa were also observed but rarely any Corynebacterium spp. were observed. Correspondingly, P. aeruginosa but not Corynebacterium spp. exhibited higher cytotoxicity to non-phagocytic HBMEC, producing more than 75% cell death in 24 h, compared to 20% cell death observed with Corynebacterium spp. Additionally, it was observed that the bacterial conditioned medium had no negative effect on A. castellanii growth. Further characterization of amoebal and bacterial interactions will assist in identifying the role of Acanthamoeba in the transmission and evolution of pathogenic bacteria.

Tigecycline inhibits proliferation of Acanthamoeba castellanii.

Acanthamoeba is an opportunistic protozoan parasite responsible for different diseases in humans, such as granulomatous amoebic encephalitis and amoebic keratitis. Tigecycline, a third-generation tetracycline antibiotic, has potential activity to treat most of the antibiotic resistant bacterial infections. The effects of tigecycline in eukaryotic cells as well as parasites are less well studied. In the present study, we tested the effects of tigecycline on trophozoites of Acanthamoeba castellanii. The inhibitory effect of tigecycline on Acanthamoeba was determined by resazurin reduction and trypan blue exclusion assays. We found that tigecycline significantly inhibited the growth of Acanthamoeba (46.4 % inhibition at the concentration of 100 μM) without affecting cell viability and induction of encystation, whereas other tetracycline groups of antibiotics such as tetracycline and doxycycline showed no inhibitory effects. Furthermore, tigecycline decreased cellular adenosine triphosphate (ATP) level by 26 % than the control and increased mitochondrial mass, suggesting mitochondrial dysfunction in tigecycline-treated cells. These findings suggest that mitochondrial dysfunction with decreased ATP production might play an important mechanism of tigecycline in suppression of Acanthamoeba proliferation.

Encystment in Acanthamoeba castellanii: A review

Differentiation of Acanthamoeba castellanii trophozoites involves massive turnover of cellular components and remodelling of organelle structure and function so as to produce a cryptobiotic cell, resistant to desiccation, heat, freezing, and chemical treatments. This review presents a summary of a decade of research on the most studied aspects of the biochemistry of this process, with emphasis on problems of biocide and drug resistances, putative new targets, molecular and cell biology of the process of encystment, and the characteristics of the encysted state. As well as the intrinsic pathogenicity of the organism towards the cornea, and the ability of related species to invade the human brain, its propensity for harbouring and transmitting pathogenic bacteria and viruses is considerable and leads to increasing concerns. The long-term survival and resistance of cysts to drugs and biocides adds another layer of complexity to the problem of their elimination.