Antimicrobial Action of Biguanides on the Viability of Acanthamoeba Cysts and Assessment of Cell Toxicity

PURPOSE. To assess dose- and concentration-dependent rates of biguanides on the viability of Acanthamoeba cysts isolated from severe ulcerative keratitis, and to correlate cysticidal activites with cytotoxic profiles in corneal and endothelial cells. METHODS. Cysticidal activities of polyhexamethylene biguanide and chlorhexidine digluconate were evaluated in the Acanthamoeba castellanii strain and clinical isolates of Acanthamoeba spp obtained from two severe and recurrent cases of ulcerative keratitis. The molecular characterization of protozoa used in the experimental assays was performed by sequencing reactions of the 18S rDNA gene. Acanthamoeba cysts were exposed at different dosages and concentrations of both biguanides; the application of double-biguanides was also evaluated. Automated cell viability assessment of cysts was performed using the trypan blue dye exclusion method. Cytotoxicity assays of biguanides were conducted using primary cultures of endothelial cells alone or in coculture with Acanthamoeba cysts. Human corneal epithelial cells were used as a comparative pattern to assess the toxicity of biguanide compounds. Cell viability was measured using both quantitative and qualitative methods. Statistical analyses were applied to the data. RESULTS. The in vitro study showed that all dosages, concentrations, and combinations of biguanides tested had a cysticidal effect on Acanthamoeba spp strains tested compared with control cultures not exposed to any antimicrobials; the difference in response was statistically significant. The use of both biguanides in combination demonstrated the best cysticidal effect. The use of isolated biguanides was associated with greater cytotoxic effects than with biguanides used in combination. Chlorhexidine digluconate used alone tended to have greater cytotoxicity than polyhexamethylene biguanide. Furthermore, the double-biguanide application had a statistically significant decrease in the deleterious effect on endothelial cells at higher dosage and concentration. Quantitative and qualitative analyses demonstrated the toxic effect of biguanide compounds on the viability of corneal epithelial cells, under single or in combination usage. CONCLUSIONS. We demonstrated that the combined use of biguanides had greater cysticidal activity than individual drug application as well as a possible protective effect on endothelial cells. The biguanide compounds tested were able to induce corneal epithelial cell death in time and concentration-independent fashions. Findings support the hypothesis concerning the cysticidal effect and the differential patterns of toxicity expressed by polyhexamethylene biguanide and chlorhexidine digluconate on the endothelial and corneal cells.