Evaluation of the effect of 2-chalcone mediated by photodynamic therapy on mono- and dual-species biofilms of Candida spp. and dermatophytes and mechanisms of interaction of microorganisms in the mixed biofilm

Various fungi may cause dermatomycoses, and dermatophytes and yeast are the most prevalent, either isolated or associated. Dermatophyte and Candida species can form biofilms associated with increased virulence, drug resistance, and, consequently, the persistence of infection. Therefore, it is necessary to intensify studies in biofilms and expand the therapeutic arsenal. The study aimed to deepen the mono and dual-species biofilms of Candida sp and dermatophytes, as well as a new antifungal agent, its toxicity, and mechanism of action. Therefore, the formation of Microsporum canis biofilms and other dermatophytes was studied. The antibiofilm capacity of 2- hydroxychalcone (2-chalcone) was evaluated in the dark and mediated by photodynamic therapy (PDT), as well as its in vitro toxicity in a cell monolayer model (2D), and a three-dimensional model (3D) using human skin keratinocyte (HaCat) and human dermal fibroblast (HDFa) lines, and in vivo in Caenorhabditis elegans and Galleria mellonella larvae. In addition, factors that promote inhibition of the interaction of dermatophytes and C. albicans and C. parapsilosis in biofilms were studied. M. canis biofilms were characterized by quantifying metabolic activity, biomass, polysaccharide structures, and extracellular matrix in RPMI 1640, BHI broth, DMEM, and HAM-F12. Susceptibility assays were carried out on clinical isolates of M. canis and reference strains of Trichophyton rubrum, T. mentagrophytes, C. albicans, and C. parapsilosis against 2-chalcone and conventional antifungals. 2- chalcone (in the dark and mediated PDT) and commercial antifungals were evaluated for their ability to inhibit planktonic cells (106 cel/ml), mono and dual-species biofilms in the initial and mature stages. Membrane sterols, reactive oxygen species (ROS) production, cell wall damage, and death mechanism were verified in cells treated with 2-chalcone in the dark and photoexcited. The effect of cell-free supernatant from T. rubrum, C. albicans or C. parapsilosis biofilms was also evaluated on the ability to inhibit biofilms and C. albicans filamentation by reducing XTT, SEM, and CM. M. canis was able to form robust biofilms in 96 hours of incubation on BHI, DMEM, and HAM-F12 media than on RPMI-1640 media. 2-chalcone inhibited initial and mature dermatophyte biofilms at concentrations greater than or equal to 31.25 mg/L. 2-chalcone PDT-mediated reduced the inhibition concentrations in planktonic cells and biofilms by 4-fold compared to 2-chalcone in the dark. The SEM and MC images showed a total collapse of the cell walls with a significant thickness reduction of the biofilms. In Candida biofilms and dual-species biofilms, 2-chalcone was less potent, although photoexcitation reduced its minimum inhibitory concentration. Dermatophyte biofilms were resistant to fluconazole and terbinafine. The same was observed in mono and dual-species biofilms against itraconazole, fluconazole and caspofungin. The 2D and 3D models of cell lines and C. elegans showed the toxicity of the 2-chalcone in dark and photoexcited at concentrations above 125 mg/L. In G. mellonella, 2-chalcone did not show toxicity even at 200 mg/kg concentrations. Treatment with 2- chalcone caused deformity in the fungal cell wall, reduced membrane sterols, and promoted ROS generation, resulting in cell death by apoptosis and necrosis. Cell-free supernatants did not reduce the metabolic activities of the biofilms; however, there was a reduction in the thickness of the biofilms. The direct interaction between the microorganisms influenced the filamentation inhibition of C. albicans in the mixed biofilms of T. rubrum and C. albicans. Therefore, this study opens doors to unravel the mechanisms of interaction between microorganisms in the mixed biofilm and points to PDT-mediated 2-chalcone as a potent therapeutic alternative for the treatment of dermatophytosis. (AU)