The Effect of Anti-Amoebic Agents and Ce6-PDT onAcanthamoeba castellaniiTrophozoites and Cysts, In Vitro

Abstract

The purpose of this study was to analyze the concentration-dependent effects of biguanides (polyhexamethylene biguanide [PHMB], chlorhexidine [CH]); diamidines (hexamidine-diisethionate [HD], propamidine-isethionate [PD], dibromopropamidinediisethionate [DD]); natamycin (NM); miltefosine (MF); povidone iodine (PVPI), and chlorin e6 PDT on Acanthamoeba trophozoites and cysts, in vitro. Methods: Strain 1BU was cultured in peptone-yeast extract-glucose medium. Trophozoites or cysts were cultured in PYG medium containing each agent at 100%, 50%, and 25% of maximum concentration for 2 hours. The percentage of dead trophozoites was determined using a non-radioactive cytotoxicity assay and trypan blue staining. Treated cysts were also maintained on non-nutrient agar Escherichia coli (E. coli) plates and observed for 3 weeks. Results: All tested drugs displayed significant cytotoxic effects on 1BU cells based on the biochemical and staining-based viability assays tested. On non-nutrient agar E. coli plates, neither trophozoites nor freshly formed cysts were observed after PHMB, PD, NM, and PVPI treatment, respectively, within 3 weeks. However, CH-, HD-, DD-, and MFtreated cysts could excyst, multiply, and encyst again. Conclusions: The off-label drugs PHMB, PD, NM, and PVPI are under in vitro conditions more effective against strain 1BU than CH, HD, DD, and MF. Our findings also suggest that the non-nutrient agar E. coli plate assay should be considered as method of choice for the in vitro analysis of the treatment efficacy of anti-amoebic agents. Translational Relevance: Ophthalmologists may optimize the treatment regime against Acanthamoeba keratitis by pre-testing the in vitro susceptibilities of the Acanthamoeba strain against drugs of interest with the non-nutrient E. coli agar plate assay.

Assessing Acanthamoeba cytotoxicity: comparison of common cell viability assays

“…While generally, the cytotoxicity measured with the LDH assay was similar to that reported for A. castellanii on HCECs in a previous study ( Sohn et al, 2019 ), we found that the LDH assay underestimated the proportion of dead cells and overestimated the proportion of living cells and thus was not reliable to determine the cytotoxic effect of Acanthamoeba on HCECs. However, the LDH assay has also been used to evaluate the activity of potentially anti-amoebic agents, because this enzyme is released into the culture medium when the cell membrane integrity is affected ( Sissons et al, 2005 ; Lorenzo-Morales et al, 2010 ; Anwar et al, 2018 ; Shi et al, 2020 ; Akbar et al, 2022 ). The results obtained in the current study corroborate the suitability of the LDH assay for evaluating Acanthamoeba viability, but not when Acanthamoeba is co-cultured with other cells.…”

Efficacy of Off-Label Anti-Amoebic Agents to Suppress Trophozoite Formation of Acanthamoeba spp. on Non-Nutrient Agar Escherichia Coli Plates

“…isolates of sequence types T4 and T6. In the process, the focus was on cysts, the dormant stage of this amoeba, which are usually less susceptible to drug treatment than the actively dividing trophozoites [8,14]. We treated the cyst suspension with each drug for 2 h, then diluted the cysts and drug suspension by 100-fold, and placed an aliquot of the diluted cyst/drug mixture onto the center of a non-nutrient agar E. coli plate, thereby transferring lower concentrations of the drug onto the test plate.…”

“…Peptone-yeast-glucose medium (PYG), Neff's constant-pH encystment medium, and Page's amoeba saline (PAS) were prepared as described in [8]. For the non-nutrient agar, 15 g of agar (Sigma-Aldrich) was mixed with 100 mL PAS and 900 mL distilled water and autoclaved at 121 • C for 15 min.…”

“…Culturing of 1BU, 3ST, 9GU, or 11DS trophozoites was conducted as described in [8]. Briefly, trophozoites were grown in tissue culture flasks containing 5 mL of PYG broth medium at 30 • C, in an airtight container.…”

“…The non-nutrient agar Escherichia coli plate assay was carried out as described in [8]. Briefly, cells of E. coli strain IM08B [11] were grown overnight on sheep blood agar plates (BD, Heidelberg, Germany) at 35 • C. Fresh grown IM08B colonies were picked with a cotton swab and suspended in PAS equivalent to a McFarland of 4.5.…”