In vitro Evaluation the Efficacy of Some New Plant Extracts and Biocides on the Viability of Acanthamoeba castellanii.

The purpose of this study was to assess the efficacy of certain plant extracts and to compare them with current biocides on the viability of Acanthamoeba castellanii cysts and trophozoites in vitro. Amoebicidal and cysticidal assays were performed against both trophozoites and cysts of Acanthamoeba castellanii (ATCC 50370). Ten plant extracts were evaluated alongside the current agents included polyhexamethylene biguanide (PHMB), octenidine and chlorhexidine digluconate. A. castellanii (ATCC 50370) was treated to serial two-fold dilutions of the test compounds and extracts in microtitre plate wells to investigate the effect on trophozoites and cysts of A. castellanii (ATCC 50370). Furthermore, the toxicity of each of the test compounds and extracts were assessed towards a mammalian cell line. Minimum trophozoite inhibitory concentration (MTIC), minimum trophozoite amoebicidal concentration (MTAC), and minimum cysticidal concentration (MCC) were used to establish A. castellanii (ATCC 50370) in vitro sensitivity. The findings of this research revealed that the biguanides PHMB, chlorhexidine, and octenidine all had excellent effectiveness against trophozoites and cysts of A. castellanii (ATCC 50370). The plant extracts testing results showed that, great activity against trophozoites and cysts ofA. castellanii (ATCC 50370) at lower concentrations. This is the first study to demonstrate that the Proskia plant extract had the lowest MCC value, which was 3.9 µg/mL. The time kill experiment confirmed this finding, as this extract reduced cysts of A. castellanii (ATCC 50370) by more than 3-log at 6 hour and by 4-log after 24 hour. The anti-amoebic efficacy of new plant extracts on the viability of A. castellanii (ATCC 50370) cysts and trophozoites was comparable to existing biocide treatments and was not toxic when tested on a mammalian cell line. This could be a promising novel Acanthamoeba treatment by using the tested plant extracts as a monotherapy against trophozoites and cysts.

Potential Use of Mycobacterium paragordonae for Antimycobacterial Drug Screening Systems.

Our recent genome-based study indicated that Mycobacterium paragordonae (Mpg) has evolved to become more adapted to an intracellular lifestyle within free-living environmental amoeba and its enhanced intracellular survival within Acanthamoeba castellanii was also proved. Here, we sought to investigate potential use of Mpg for antimycobacterial drug screening systems. Our data showed that Mpg is more susceptible to various antibiotics compared to the close species M. marinum (Mmar) and M. gordonae, further supporting its intracellular lifestyle in environments, which would explain its protection from environmental insults. In addition, we developed two bacterial whole-cell-based drug screening systems using a recombinant Mpg stain harboring a luciferase reporter vector (rMpg-LuxG13): one for direct application to rMpg-LuxG13 and the other for drug screening via the interaction of rMpg-LuxG13 with A. castellanii. Direct application to rMpg-LuxG13 showed lower inhibitory concentration 50 (IC50) values of rifampin, isoniazid, clarithromycin, and ciprofloxacin against Mpg compared to Mmar. Application of drug screening system via the interaction of rMpg-LuxG13 with A. castellanii also exhibited lower IC50 values for rifampin against Mpg compared to Mmar. In conclusion, our data indicate that Mpg is more susceptible to various antibiotics than other strains. In addition, our data also demonstrate the feasibility of two whole cell-based drug screening systems using rMpg-LuxG13 strain for the discovery of novel anti-mycobacterial drugs.

Cinnamic acid and lactobionic acid based nanoformulations as a potential antiamoebic therapeutics.

Acanthamoeba castellanii causes granulomatous amoebic encephalitis, an uncommon but severe brain infection and sight-threatening Acanthamoeba keratitis. Most of the currently used anti-amoebic treatments are not always effective, due to persistence of the cyst stage, and recurrence can occur. Here in this study we synthesize cinnamic acid and lactobionic acid-based magnetic nanoparticles (MNPs) using co-precipitation technique. These nanoformulations were characterized by Fourier transform infrared spectroscopy and Atomic form microscopy. The drugs alone (Hesperidin, Curcumin and Amphotericin B), magnetic NPs alone, and drug-loaded nano-formulations were evaluated at a concentration of 100 µg/mL for antiamoebic activity against a clinical isolate of A. castellanii. Amoebicidal assays revealed that drugs and conjugation of drugs and NPs further enhanced amoebicidal effects of drug-loaded nanoformulations. Drugs and drug-loaded nanoformulations inhibited both encystation and excystation of amoebae. In addition, drugs and drug-loaded nanoformulations inhibited parasite binding capability to the host cells. Neither drugs nor drug-loaded nanoformulations showed cytotoxic effects against host cells and considerably reduced parasite-mediated host cell death. Overall, these findings imply that conjugation of medically approved drugs with MNPs produce potent anti-Acanthamoebic effects, which could eventually lead to the development of therapeutic medications.

Camellia sinensis solvent extract, epigallocatechin gallate and caffeine confer trophocidal and cysticidal effects against Acanthamoeba castellanii.

We examined the anti-acanthamoebic efficacy of green tea Camellia sinensis solvent extract (SE) or its chemical constituents against Acanthamoeba castellanii by using anti-trophozoite, anti-encystation, and anti-excystation assays. C. sinensis SE (625-5000 µg/mL) inhibited trophozoite replication within 24-72 h. C. sinensis SE exhibited a dose-dependent inhibition of encystation, with a marked cysticidal activity at 2500-5000 µg/mL. Two constituents of C. sinensis, namely epigallocatechin-3-gallate and caffeine, at 100 µM and 200 µM respectively, significantly inhibited both trophozoite replication and encystation. Cytotoxicity analysis showed that 156.25-2500 µg/mL of SE was not toxic to human corneal epithelial cells, while up to 625 µg/mL was not toxic to Madin-Darby canine kidney cells. This study shows the anti-acanthamoebic potential of C. sinensis SE against A. castellanii trophozoites and cysts. Pre-clinical studies are required to elucidate the in vivo efficacy and safety of C. sinensis SE.

In vitro anti-Acanthamoeba activity of flavonoid glycosides isolated from Delphinium gracile, D. staphisagria, Consolida oliveriana and Aconitum napellus.

Acanthamoeba spp. are widely distributed in the environment and cause serious infections in humans. Treatment of Acanthamoeba infections is very challenging and not always effective which requires the development of more efficient drugs against Acanthamoeba spp. The purpose of the present study was to test medicinal plants that may be useful in the treatment of Acanthamoeba spp. Here we evaluated the trophozoital and cysticidal activity of 13 flavonoid glycosides isolated from Delphinium gracile, D. staphisagria, Consolida oliveriana and from Aconitum napellus subsp. Lusitanicum against the amoeba Acanthamoeba castellanii. AlamarBlue Assay Reagent® was used to determine the activity against trophozoites of A. castellanii, and cytotoxic using Vero cells. Cysticidal activity was assessed on treated cysts by light microscopy using a Neubauer chamber to quantify cysts and trophozoites. Flavonoids 1, 2, 3 and 4 showed higher trophozoital activity and selectivity indexes than the reference drug chlorhexidine digluconate. In addition, flavonoid 2 showed 100% cysticidal activity at a concentration of 50 µm, lower than those of the reference drug and flavonoid 3 (100 µm). These results suggest that flavonoids 2 and 3 might be used for the development of novel therapeutic approaches against Acanthamoeba infections after satisfactory in vivo evaluations.

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.

Rose Bengal-Mediated Photodynamic Antimicrobial Treatment of Acanthamoeba Keratitis.

Purpose: To evaluate the in vivo efficacy of rose bengal (RB)-mediated photodynamic antimicrobial therapy (PDAT) for treatment of Acanthamoeba castellanii keratitis (AK). Materials and Methods: An animal (rabbit) AK model was successfully achieved via intrastromal inoculation of a suspension of A. castellanii cells and trophozoites. Prior to RB-PDAT (pre-treatment, day-5), the severity of the induced corneal infection was graded numerically for epithelial defects, stromal edema, neovascularity, and stromal opacity/infiltration. The right eyes of rabbits (n = 18) were divided equally into three groups (n = 6/group): control (no treatment); 0.1% RB+518 nm irradiation (5.4 J/cm2); and 0.2% RB+518 nm irradiation (5.4 J/cm2). On post-treatment day-5, animals were euthanized, after which corneal buttons were excised and submitted for real-time polymerase chain reaction (RT-PCR) analysis. Results: Post-treatment clinical scores of the 0.1 and 0.2% RB groups indicated significant improvement compared to control group scores (pre-treatment clinical scores; 5.17 ± 0.98, 7.50 ± 0.62, and 6.17 ± 0.70 and post-treatment clinical scores; 4.50 ± 0.56, (p = .043), 3.50 ± 0.99 (p = .039), 6.83 ± 1.66 (p = .34), respectively). RT-PCR analysis revealed that the mean cycle threshold (Ct) values were significantly higher in treated-group corneas compared to control-group corneas, with no significant differences between treated-groups (Mean Ct values; 34.33, 34.5, and 29.67 for 0.1 and 0.2% RB, and control groups). There was a statistically significant negative correlation between post-treatment clinical scores and Ct values (r = -0.474, p-value 0.047). Conclusions: Our results demonstrate that RB-PDAT is effective in decreasing the parasitic load and clinical severity of AK.

Amebicidal effects of fenugreek (Trigonella foenum-graecum) against Acanthamoeba cysts.

Trigonella foenum-graecum L. (TF) is known to the public as a chest emollient, mucous expectorant, laxative and is used to prevent maturation of boils and diabetes since ancient times. In this study, we aimed to determine the amebicidal effects against Acanthamoeba cysts. Plant extracts were prepared at concentrations of 1, 2, 4, 8, 16, and 32 mg/ml and were placed in a hemocytometer with cell counts 22 × 106 cell/ml. The fatty acid profiles of TF seeds were determined. Standard Acanthamoeba cysts were added and incubated at 25°C. The viability of the parasite was checked and recorded at hours 3, 24, 48, 72, 96, and 102. The values of lethal concentration doses (LD50 and LD90) were calculated using probit analysis. This study revealed that T. foenum-graecum prevented proliferation of the parasite at certain times. However, further for in vivo and controlled experimental studies are needed in order to find out how to use this plant as medication.

Amoebicidal and Amoebistatic Effects of Artemisia argyi Methanolic Extracts on Acanthamoeba castellanii Trophozoites and Cysts.

PURPOSE : The present study aimed to investigate the amoebicidal and amoebistatic efects of Artemisia argyi leaf methanolic extract by testing the effects on trophozoites and on cysts. We also determined cytotoxic effect, enzymatic and non-enzymatic antioxidant activities, total phenolic, lavonoid and antioxidative contents of A. argyi. METHODS: A. argyi was harvested from various geographic sites in Ordu province in Turkey. The fresh leaves were subjected to methanolic extraction. In 100 µl culture, different concentrations of A. argyi methanolic extract (in quantities from 1.2, 2.3, 4.7, 9.4, 18.7, 37.4, 74.8 mg/ml) and the same volume of trophozoite/cyst suspension were mixed for the determination of the amoebicidal activity of the plant extract. Human bronchial epithelial cells were treated with the same concentrations of Artemisia extracts to determine cytotoxic potential. RESULTS: Total phenolic and lavonoid contents of the extract were calculated as 261 mg gallic acid/g dry extract and 29 mg quercetin/g dry extract, respectively. Total antioxidant activity was also calculated as 367 mg ascorbic acid/g dry extract. The growth of trophozoites stopped in A. argyi methanolic extract with 50% inhibitory concentrations (IC50)/8 h for 37.4 mg/ ml and 74.8 mg/ml extract solution and had stronger amoebicidal activity on the cysts with IC50/72 h. Artemisia showed stronger inhibitory effects on bronchial epithelial cells at the concentrations of 9.4, 18.7, 37.4 and 74.8 mg/ml. CONCLUSION: The study indicated that A. argyi leaf extract has cytotoxic and anti-amoebic activities.

Essential oil composition and anti Acanthamoeba studies of Teucrium ramosissimum.

The aim of the present study was to evaluate the chemical composition of the essential oil obtained from the aerial parts of T. ramosissimum by hydrodistillation and to investigate their anti-Acanthamoeba activity. Identification and quantification were realized by gas chromatography-mass spectrometry (GC-MS) and gas chromatography with flame ionization detection by (GC-FID). Sixty-eight compounds representing 97.78% of the essential oil were identified, of which δ-cadinene (18.63%), δ-cadinol (18.70%), ß-eudesmol (12.13%), γ-gurjunene (4.34%) and 8-cedrene (3.99%) were the main compounds. This essential oil contained a complex mixture consisting mainly on sesquiterpenes (80.62%) and monoterpene fractions (14.34%). The findings of the anti-Acanthamoeba assay indicate that T. ramosissimum essential oil have a good activity with an IC50 = 25.73 ± 0.75 µg/mL.

Identification and Characterization of Protein Arginine Methyltransferase 1 in Acanthamoeba castellanii

Protein arginine methyltransferase (PRMT) is an important epigenetic regulator in eukaryotic cells. During encystation, an essential process for Acanthamoeba survival, the expression of a lot of genes involved in the encystation process has to be regulated in order to be induced or inhibited. However, the regulation mechanism of these genes is yet unknown. In this study, the full-length 1,059 bp cDNA sequence of Acanthamoeba castellanii PRMT1 (AcPRMT1) was cloned for the first time. The AcPRMT1 protein comprised of 352 amino acids with a SAM-dependent methyltransferase PRMT-type domain. The expression level of AcPRMT1 was highly increased during encystation of A. castellanii. The EGFP-AcPRMT1 fusion protein was distributed over the cytoplasm, but it was mainly localized in the nucleus of Acanthamoeba. Knock down of AcPRMT1 by synthetic siRNA with a complementary sequence failed to form mature cysts. These findings suggested that AcPRMT1 plays a critical role in the regulation of encystation of A. castellanii. The target gene of AcPRMT1 regulation and the detailed mechanisms need to be investigated by further studies.

Lethal effects of Helianthemum lippii (L.) on Acanthamoeba castellanii cysts in vitro.

Acanthamoeba spp. commonly cause Acanthamoeba keratitis which is typically associated with the wear of contact lenses. Therefore, finding an economic, efficient, and safe therapy of natural origin is of outmost importance. This study examined the in vitro lethal potential of ethyl acetate and methanol extracts of Helianthemum lippii (L.) (sun roses) against Acanthamoeba castellanii cysts isolated from patients with amoebic keratitis. Both extracts proved to be potent as regard to their lethal effects on A. castellanii cysts with comparable results to chlorhexidine. The ethyl acetate was more promising with cumulative lethality. It showed a highly significant lethal percentage along the duration of treatment. The analysis of the more potent ethyl acetate extract revealed the presence of 2.96 mg/100 g of total phenolics, 0.289 mg/100 ml of total flavonoids and 37 mg/100 mg of total tannins which highlighted their phytomedicinal role.