Recently published findings showed that curcumin-mediated Photodynamic Therapy (PDT) is a promising alternative to inactivate Candida species. Given the importance of investigating the effects of this potential photosensitizer, this research project propose to conduct two studies, which will evaluate the effect of PDT mediated by curcumin on the expression of virulence factors of Candida species, and the influence of PDT in the susceptibility of Candida to fluconazole and amphotericin B. Reference strains of C. albicans, C. glabrata, C. tropicalis and C. krusei will be used in Study I. Standardized cell suspensions of each strain will be exposed to sublethal curcumin concentrations and light fluencies, thus allowing the survival of microorganisms after PDT. Then, samples of the fungal cells will be used to assess the expression of different virulence factors: adhesion to epithelial cells, adhesion and biofilm formation on abiotic surfaces, enzymes secretion (protease and phospholipase) and production of filamentous forms of growth. The control group will be consisted of Candida suspensions not exposed to either curcumin or light. The aim of Study II will be to determine whether a pretreatment with PDT would affect subsequent antifungal susceptibility of Candida cells and biofilms. The same reference strains described in Study I will be used, but fluconazole-resistant strains of C. albicans, C. glabrata and C. krusei, and amphotericin B-resistant strains of C. albicans and C. tropicalis will be also included. In the first phase of experiments, standardized cell suspensions will be obtained and then exposed to curcumin-PDT. Then, the minimum inhibitory concentration (MIC), in ¼g/ml, of the antifungals amphotericin B and fluconazole will be determined in the samples of Candida previously exposed to PDT. The control group will be consisted of Candida suspensions not exposed to either curcumin or light. Also, the viability of Candida suspensions exposed or not exposed to PDT, will be assessed by colony counts and flow cytometry using propidium iodide (PI). The second phase of Study II will evaluate the fluconazole and amphotericin B action on PDT-treated biofilms. For this reason, the same strains described above will be used for in vitro biofilm growth. The mature biofilms will be exposed to the antifungal drugs for a period of 24 hours and then the metabolic activity will be analyzed by means of XTT reduction assay. In addition, the biofilm structure will be assessed with confocal microscopy (BacLight Live/Dead viability kit) and matrix composition will be evaluated in terms of protein and carbohydrate content (extracellular polymeric substances).
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