Biofilme de dermatófitos in vitro e ex vivo: formação, arquitetura e sensibilidade

Abstract

The microorganisms when presented in biofilm express properties different from when they are in the planktonic form. One of these properties is a higher resistance to antimicrobial agents. The aim of this study was to evaluate the formation of dermatophytes’ biofilms in in vitro and ex vivo assay performed in polystyrene coverslip and nail fragments, respectively, and their susceptibility to three antifungal agents (itraconazole, voriconazole and griseofulvin) in planktonics and biofilm forms. To this end, at first, they were used 26 strains isolated from clinical specimens of human and animal: Trichophyton rubrum (4) Trichophyton tonsurans (6), Trichophyton mentagrophytes (3), Microsporum gypseum (3) e Microsporum canis (10). The biofilms were initially formed in microdilution plates, and the strains were classified by the amount of biomass as non producers (2/26), weak (7/26), moderate (1/26) and strong biofilm producers (16/26). Then, strains, strong producers of biofilms, were chosen, one of each species, to biofilm formation in coverslip Thermanox® and nail fragments for later analysis by optical, confocal and scanning electron microscopy. The analyzes demonstrated that all species formed biofilms in vitro and ex vivo, with different density and architecture. M. gypseum and M. canis produced biofilms with higher and lower amounts of biomass, respectively. At a second step, according to the recommendations of the CLSI M38-A2 protocol, the minimum inhibitory concentration (MIC) values for the three antifungal agents against these 26 strains were determined, in the planktonic form. MICs values ranged from 0.00195-0.25 μg/ml for itraconazol, 0.00195-0.125 for voriconazole and <0.0039-4 μg / ml for griseofulvin. Subsequently, biofilms were formed, in microdilution plates and treated with the same drugs, in MIC, 10 x MIC and 50 x MIC values. The cell viability of the biofilms was quantified using the XTT colorimetric assay. In order to significantly reduce the metabolic activity of biofilms (P <0.05), 50 x MIC was required, for the three drugs. This study may contribute to improve the understanding of the pathophysiology of the microorganism and tolerance or resistance to treatment.