Efeito in vitro da proteinase K na matriz do biofilme de fungos dermatofíticos: uma análise das interações com terbinafina e griseofulvina

Abstract

The capacity of dermatophytes to form biofilm is virulence factor that potentiate the infection. The polymeric extracellular matrix whose composition, such as protein, differs among microorganisms, provides a microenvironment capable of increasing resistance to antifungal agents. The aim of this study was the analysis of proteinase K in the matrix of mature biofilms of dermatophytes, as well as to investigate the interaction of proteinase K with terbinafine or griseofulvin. Initially, the sensitivity test of planktonic cells was performed using the broth microdilution method with 14 isolated strains of animal and human Microsporum canis (1), Trichophyton tonsurans (5), Trichophyton mentagrophytes (4), Epidermophyton floccosum (1) Trichophyton rubrum (1). Subsequently, on microplates, strains strong biofilms forms were tested with proteinase K and with antifungal agents, being evaluated by quantification of metabolic activity and biomass. Afterwards, proteinase K and terbinafine or griseofulvin were combined against biofilms of M. canis (2) and T. tonsurans (2), therefore, they were evaluated by means of metabolic activity. The biofilm architecture was analyzed by scanning and confocal electron microscopy. The minimum inhibitory concentration (MIC) ranges against the planktonic form of dermatophytes were >250 µg ml-1 for proteinase K, 0.0078 - 0.25 µg ml -1 for terbinafine, and 0.125 - 0.5 µg ml-1 for griseofulvin. As for mature biofilms, proteinase K at 32 µg ml-1 (P <0.0001) reduced metabolic activity by 39% and biomass by 37.62%, while terbinafine at 128 µg ml-1 (P < 0.0001) reduced metabolic activity by 39.9% and biomass by 43.5%, whereas griseofulvin at 128 µg ml-1 (P <0.001) reduced metabolic activity by 51% and biomass by 53.6%. The association of proteinase K with terbinafine or griseofulvin showed a synergistic effect (P<0.05) against biofilms of M. canis and T. tonsurans. Microscopy images showed reduced biofilm when treated with proteinase K and when combined with antifungal agents. Thus, this study demonstrates the ability of proteinase K to reduce the metabolic activity and biomass of dermatophyte biofilms, as well as its synergistic potential in association with terbinafine and griseofulvin.