Evaluation of the efficacy of DNase associated with photodynamic therapy against fluconazole resistant Candida albicans biofilm

Abstract

Studies have shown that antimicrobial photodynamic therapy (aPDT) promotes complete inactivation of microorganisms in suspension, however, the complete inactivation of biofilms has not yet been observed. The resistance of Candida spp. biofilm is multifactorial and has been associated with factors such as the protective effect of the extracellular matrix of biofilm (MEC) performed by ²-glucans, which bind to fluconazole and amphotericin B, preventing the drugs penetration into the biofilm. The MEC biofilm also hinders the photosensitizing penetration agent and other drugs. Another important aspect is that Candida species resistant to fluconazole have also been shown to be more resistant to aPDT. Based on that, this study aims to evaluate the efficacy of DNase enzyme associated with aPDT on biofilms of clinical isolates of Candida albicans resistant to fluconazole. Biofilms will be formed from resistant strains of C. albicans [ATCC 96901, and two clinical isolates (R10 and R15)]. After 48 h of biofilm formation these strains were will be treated with DNase and then exposed to aPDT (incubated with Photodithazine® for 20 min and exposed to LED light (660 nm, 37.5 J/cm2). The following methods will be used to evaluate the treatments effectiveness: colony-forming unit (CFU)/mL, dry weight analysis, protein quantification by Bradford method, analysis of water-soluble polysaccharides (WSP), analysis of alkali soluble polysaccharides (ASP) and analysis of eDNA. Besides that, the biofilm structure will be evaluated through Confocal Laser Microscopy. The data obtained will be submitted to the most adequate statistical analysis for each experimental condition. (AU)