Mode-of-action evaluation of the antimycotic effect of a tetracycline-chloramphenicol-colistimethate sodium fixed-dose combination

Purpose. Previous our studies showed that the a commercially available ophthalmic product, as fixed antibiotic combination (ColbiocinTM), containing tetracycline, chloramphenicol and colistimethate sodium, had a good efficacy against Candida spp., including resistant strains (Marino et al., ARVO 2015). The aim of this work was to study the mode-of-action of this antibacterial product against yeasts. Methods. C. albicans ATCC and clinical isolated treated with sub-MIC concentrations of Colbiocin eye drop or with antibiotics of the formulation, alone and in combination each other, were examined by propidium iodide and MitoTracker red staining to determine cell permeability and mitochondrial function, respectively. Observations under the fluorescence microscope were performed. Results. Microscopic examination using propidium staining demonstrated that the Candida cells treated with colistimethate sodium were stained red because they lost in cell membrane integrity. Infact, propidium iodide a red-fluorescent nuclear stain, is a membrane impermeant dye that is generally excluded from yeast viable cells. Microscopic examination using MitoTracker demonstrated that Candida cells treated with tetracycline or chloramphenicol showed morphological changes and alteration of mitochondrial mass. This last fluorescent dye, labels mitochondria within live cells utilizing the mitochondrial membrane potential. However, once incorporated in the mitochondria, it can chemically link to thiol groups and will leave the mitochondria when the membrane potential decreases as a result of cell death. The Candida cells treated with the fixed combination Colbiocin and after stained with propidium iodide demonstrated membrane permeabilization, while changes in mithocondrial structure were evident when the MitoTracker dye was used. Conclusions. The additive effect of the antibiotics present in Colbiocin fixed dose combination against C. albicans cells is explained as follow: the colistimethate sodium binds with anionic lipids disrupting membrane integrity thus allowing to tetracycline and chloramphenicol to penetrate through the lipid bilayer and promote mitochondrial dysfunction. These new results on the mode-of-action, support our previously published data and the use of the fixed combination ColbiocinTM in the treatment of yeast infections. .