Avaliação in vitro da atividade antifúngica do midazolam frente a isolados de Candida spp. resistentes ao fluconazol, sua atividade contra biofilme formado e investigação do provável mecanismo de ação

Abstract

The occurence of invasive candidiasis in tertiary hospitals has increased significantly in recent decades, covering approximately 80% of nosocomial fungal infections. One of the factors that contribute to this percentage is the appearance of the resistance to antifungals, which can be influenced by the ability of some species of Candida spp. to form biofilms and also by the reduced therapeutic arsenal, makes it necessary to search for new alternatives to overcome this limitation. The objective of the work was to evaluate the antifungal activity of midazolam isolated and in association with azolics in relation to clinical isolates of Candida spp. resistant to fluconazole in planktonic form and in biofilm. The microdilution broth technique described by the Clinical and Laboratory Standard Institute (CLSI, M27-A3) was used to determine the Minimum Inhibitory Concentration (MIC). Flow cytometry tests were also performed to investigate the probable mechanism of action, evaluation of DNA damage by the comet test and cytotoxicity test on L929 murine fibroblasts. Midazolam showed MIC ranging from 125 to 250 μg/mL. Midazolam showed no cytotoxicity when evaluated at concentrations of 31.5 to 500 μg/mL. It showed significant reduction in cellular viability of Candida albicans biofilm by 500 μg/mL, Candida tropicalis by 1000 μg/mL and Candida parapsilosis by 125 μg/mL. It was also found that midazolam promotes decreased cell density, damage to the mitochondrial membrane and that it provided phosphatidylserine externalization but was not able to produce reactive oxygen species and cause DNA damage. The interaction of midazolam and Candida spp. biofilm was also observed through scanning electron microscopy (SEM), where the treated cells presented alteration in their appearance, which may have been caused by the loss of rigidity of the fungal cell wall. Therefore, midazolam proved to be an antifungal potential against the isolates tested in this study.