Atividade de um biossurfactante lipopeptídico produzido por Bacillus subtilis sobre células planctônicas e biofilmes de Trichosporon spp

Abstract

In recent years, studies have given relevance to Trichosporon, a fungal genus that includes emerging opportunistic pathogenic species that infect patients presenting hematological malignancies. Among the virulence factors expressed by these fungal species, biofilm formation stands out, a cell community that presents tolerance to antifungal agents for therapeutic use. New effective therapeutic measures become necessary. Biosurfactants showed to be a viable alternative as they present low toxicity and reduction of the surface tension of the medium. Thus, the present study aimed to investigate the effect of a biosurfactant (TIM96) produced by Bacillus subtilis on planktonic and sessile cells of Trichosporon spp. For this, susceptibility of clinical strains of T. asahii (n = 4) and T. inkin (n = 6) was evaluated by the broth microdilution test. Susceptibility of the strains against TIM96, amphotericin B (AMB), fluconazole (FLC) and voriconazole (VCZ) was evaluated, as well as the interaction between the surfactant and the antifungal agents. Time-kill curve assay was evaluated with fungal cells exposed to TIM96 through analysis of the colony forming units (CFU). The effect of TIM96 on ergosterol content, damage to fungal membranes and cell hydrophobicity in planktonic cells were also evaluated. The inhibitory activity of TIM96 against biofilm formation as well as mature biofilms was evaluated using polystyrene microplate and spectrophotometric analyzes of cell viability and biomass were performed, as well as confocal microscopy. Finally, the toxicity of TIM96 was evaluated in murine macrophages culture. MIC values of TIM96 ranged from 78.125 to 312.5 μg/mL; for antifungals, MIC values ranged from 0.125 to 2 μg/mL for AMB; 1 to 8 μg/mL for FLC and 0,0312 a 0,125 µg/mL for VCZ. When tested in combination with antifungals, pharmacological indifference and antagonism were observed. Time-kill curve revealed that the decline in the number of colonies occurred after 6 hours of contact with the surfactant, with total death occurring after 24 hours. TIM96 at subinhibitory concentrations reduced the cell ergosterol content, also altering membrane permeability and surface hydrophobicity of planktonic cells of Trichosporon. Incubation with TIM96 in 10XMIC reduced cell adhesion up to 96.89%, interfering with the formation of biofilms. This concentration also interfered with mature biofilms with a reduction of metabolic activity up to 99.2%. Concentrations of TIM96 ranging from 78,125 to 5000 μg/mL showed toxicity to murine macrophages. The results open perspectives for the discovery of new antifungal strategies