Extracellular matrix of susceptible and fluconazole-resistant Candida albicans and Candida glabrata biofilms: characterization, action of hydrolytic enzymes and efficacy of the combination of enzymes to Photodynamic Therapy.

Abstract

Infections caused by Candida spp. are often associated with biofilm formation, a complex microstructure of cells adhered to a surface and embedded within an extracellular matrix (ECM). The Candida spp biofilms resistance is multifactorial and is associated with some factors, such as the protective effect of the biofilm ECM performed by beta-glucans, which bind to fluconazole and amphotericin B, preventing the penetration of these drugs into the biofilm. Besides the protective effect of the ECM performed by beta-glucans, it has been shown that extracellular DNA (eDNA) also contributes to the structural integrity of biofilm. Studies evaluating the efficacy of PDT suggest that the therapy is effective and promotes complete inactivation of microorganisms in suspension, however, complete inactivation of the microrganisms organized in biofilms has not yet been observed. Probably, this is due to the protective effect of the biofilm ECM, which hinders the action of the photosensitizing agent. Another important aspect is that the antifungal resistant species also have shown to be more resistant to PDT. Therefore, this project aims to initially conduct a comparative evaluation of biofilms ECM of strains of Candida albicans and Candida glabrata susceptible and resistant to fluconazole through colony count, analysis of dry weight, protein quantification by the Bradford method, analysis of water soluble polysaccharides (WSP), analysis of alkali-soluble polysaccharides (ASP) and eDNA analysis. Then, will be determined the best combination of hydrolytic enzymes that would allow the degradation of ECM components of these biofilms through its association with DNase, Dextranase or beta-glucanase enzymes. The effect of the enzymes combination on the biofilms will be measured using the same methodology. Furthermore, the structure of biofilms after different periods of incubation with the enzymes will be assessed by Scanning Electron Microscopy (SEM) and Confocal Laser Microscopy. From these results will be checked if the action of photodynamic therapy mediated by photosensitizers Curcumin and Photodithazine® would be favored when associated with these enzymes. To this end, the following tests will be performed: colony count, analysis of dry weight, protein quantification by the Bradford method, analysis of water-soluble polysaccharides (WSP), analysis alkali soluble polysaccharides (ASP), analysis of eDNA, SEM and confocal laser microscopy. The data obtained will be submitted to the most appropriate statistical analyzes for each experimental condition.