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

Year: 2022
Type: dissertation
Abstract: Trichosporon comprises a fungal genus whose species have been considered emerging \npathogens causing invasive infections in humans. The infectious process is established mainly \nin immunocompromised patients, in which fungi find adequate conditions to express several \nvirulence factors, such as exoenzymes and biofilms. In addition, these agents are highly tolerant \nto therapeutic antifungal agents, contributing to an unfavorable prognosis in many patients. \nAlthough the mechanisms of resistance in Trichosporon are not fully understood, experimental \nevidence has suggested the involvement of efflux pumps in azole tolerance. It is known that \npersister cells within the biofilm also contribute to microbial resistance, however its occurrence \nhas not yet been described in Trichosporon. The objective of the present research was to \ninvestigate the formation of persister cells in biofilms of Trichosporon asahii and T. inkin, as \nwell as the in vitro effect of promethazine (PMZ) - an inhibitor of efflux pumps - against \nplanktonic and sessile cells, as well as on the formation of persister cells in Trichosporon. The \nproduction of persister cells was evaluated by exposing mature biofilms derived from clinical \nstrains of T. asahii (n=5) and T. inkin (n=7) to amphotericin B (AMB; 100 μg/ml) and \nsubsequent viable cell count. Persister cells recovered from biofilms were evaluated for \nsusceptibility to AMB and the ability to form new biofilms, which were studied for their \nstructural aspects and susceptibility to AMB. In the second part of the work, the activity of \nPMZ in strains of T. asahii (n=5) and T. inkin (n=7) was investigated. For planktonic cells, the \nbroth microdilution test was performed with determination of the minimum inhibitory \nconcentration (MIC); for biofilms, the effect of PMZ was investigated regarding metabolic \nactivity, biomass, carbohydrate and protein content, ultrastructural aspects and inhibition of \npersister cells formation. Additionally, the interaction of PMZ with AMB and fluconazole \n(FLC) in planktonic cells and biofilms of T. asahii and T. inkin was studied. The inhibition of \ncellular efflux activity by PMZ was confirmed in an assay using ethidium bromide as a \nsubstrate. Persister cells were detected in the biofilms of all strains studied, showing tolerance \nto high doses of AMB. Persister cells were also able to generate new biofilms, however these \nhad reduced metabolic activity, biomass and susceptibility to AMB compared to the control \nbiofilm. PMZ completely inhibited the growth of planktonic cells of T. asahii and T. inkin at \nconcentrations ranging from 32 to 256 μg/ml. During the adhesion and development phases of \nbiofilms, as well as in mature biofilms, there was a significant reduction in metabolic activity \nand biomass at the concentrations tested (MIC and 10x planktonic MIC). In the drug interaction \ntest, it was observed that PMZ potentiated the action of AMB and FLC, in vitro, both in \nplanktonic cells and in biofilms. PMZ affected the morphology of Trichosporon. and altered \nthe carbohydrate and protein content of these communities. Additionally, mature biofilms preexposed to sub-inhibitory doses of PMZ produced fewer persister cells. The present study \ndescribed, for the first time, the formation of persister cells in biofilms of Trichosporon. \nFurthermore, the inhibitory effect of PMZ on planktonic and sessile cells of Trichosporon was \nshown, as well as the changes induced by sub-inhibitory doses in biofilms. The results indicate \nthat efflux inhibition is a promising strategy for the control of Trichosporon.
Language: English
Field: Medicine
Open Access status: green
Funders: Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Ministério da Ciência, Tecnologia e Inovação, Conselho Nacional de Desenvolvimento Científico e Tecnológico, Centro de Tecnologias Estratégicas do Nordeste, Ministério da Ciência, Tecnologia, Inovações e Comunicações, Sistema de Laboratórios em Nanotecnologias, Universidade Federal de Viçosa
OpenAlex ID: W7008046463
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