Aspectos funcionais de biofilmes de Trichosporon: detecção de células persistentes e atividade de efluxo em cepas clínicas de Trichosporon asahii e tT inkin

Abstract

Trichosporon comprises a fungal genus whose species have been considered emerging pathogens causing invasive infections in humans. The infectious process is established mainly in immunocompromised patients, in which fungi find adequate conditions to express several virulence factors, such as exoenzymes and biofilms. In addition, these agents are highly tolerant to therapeutic antifungal agents, contributing to an unfavorable prognosis in many patients. Although the mechanisms of resistance in Trichosporon are not fully understood, experimental evidence has suggested the involvement of efflux pumps in azole tolerance. It is known that persister cells within the biofilm also contribute to microbial resistance, however its occurrence has not yet been described in Trichosporon. The objective of the present research was to investigate the formation of persister cells in biofilms of Trichosporon asahii and T. inkin, as well as the in vitro effect of promethazine (PMZ) - an inhibitor of efflux pumps - against planktonic and sessile cells, as well as on the formation of persister cells in Trichosporon. The production of persister cells was evaluated by exposing mature biofilms derived from clinical strains of T. asahii (n=5) and T. inkin (n=7) to amphotericin B (AMB; 100 μg/ml) and subsequent viable cell count. Persister cells recovered from biofilms were evaluated for susceptibility to AMB and the ability to form new biofilms, which were studied for their structural aspects and susceptibility to AMB. In the second part of the work, the activity of PMZ in strains of T. asahii (n=5) and T. inkin (n=7) was investigated. For planktonic cells, the broth microdilution test was performed with determination of the minimum inhibitory concentration (MIC); for biofilms, the effect of PMZ was investigated regarding metabolic activity, biomass, carbohydrate and protein content, ultrastructural aspects and inhibition of persister cells formation. Additionally, the interaction of PMZ with AMB and fluconazole (FLC) in planktonic cells and biofilms of T. asahii and T. inkin was studied. The inhibition of cellular efflux activity by PMZ was confirmed in an assay using ethidium bromide as a substrate. Persister cells were detected in the biofilms of all strains studied, showing tolerance to high doses of AMB. Persister cells were also able to generate new biofilms, however these had reduced metabolic activity, biomass and susceptibility to AMB compared to the control biofilm. PMZ completely inhibited the growth of planktonic cells of T. asahii and T. inkin at concentrations ranging from 32 to 256 μg/ml. During the adhesion and development phases of biofilms, as well as in mature biofilms, there was a significant reduction in metabolic activity and biomass at the concentrations tested (MIC and 10x planktonic MIC). In the drug interaction test, it was observed that PMZ potentiated the action of AMB and FLC, in vitro, both in planktonic cells and in biofilms. PMZ affected the morphology of Trichosporon. and altered the carbohydrate and protein content of these communities. Additionally, mature biofilms preexposed to sub-inhibitory doses of PMZ produced fewer persister cells. The present study described, for the first time, the formation of persister cells in biofilms of Trichosporon. Furthermore, the inhibitory effect of PMZ on planktonic and sessile cells of Trichosporon was shown, as well as the changes induced by sub-inhibitory doses in biofilms. The results indicate that efflux inhibition is a promising strategy for the control of Trichosporon.