| Congresso Brasileiro de Microbiologia 2023 | Resumo: 184-1 | ||||
Resumo:Infections caused by Candida spp. have increased significantly. Biofilm production is an important virulence factor of these yeasts and increases their resistance to antimicrobials and their persistence in the environment. Therefore, discovering new antifungal agents is urgent. Several natural products have relevant biological properties; for example, rottlerin, a natural polyphenol extracted from Mallotus philippensis, displays various pharmacological activities. This study aims to evaluate the in vitro antibiofilm activity of rottlerin against Candida species. C. albicans (ATCC 90028), C. auris (clinical isolate), and C. dubliniensis (ATCC MYA-646) strains were used in the study. Inhibition of biofilm production was evaluated in terms of biomass and cell viability. To evaluate biofilm inhibition, the biofilm was formed in microplate wells and stained with 0.1% crystal violet. Reading was performed at 595 nm on a microplate reader, and the Minimum Inhibitory Concentration of Biofilm (MICB50), defined as the lowest concentration of the sample capable of inhibiting biofilm formation by at least 50%, was determined. To evaluate cell viability, the tetrazole salt 2,3-bis (2-methoxy- 4-nitro-5-sulfophenyl)-2Htetrazolium-5-carboxanilide (MTT) was pipetted into each well. Reading was performed at 490 nm on a microplate reader, and the concentration capable of inhibiting cell viability of the biofilm by 50% (IC50) was determined. In addition, Scanning Electron Microscopy (SEM) analysis was performed in 24-well plates containing biofilms formed in 0.3-mm-diameter sterile PVC coverslips, and reading was carried out under a Tescan scanning electron microscope. The MICB50 values ranged from 15.62 to 250 µg/mL. The lowest MICB50 value was observed against C. auris (clinical isolate), and biomass decreased significantly at concentrations above MICB50. Regarding cell viability inhibition, IC50 values ranged from 2.24 to 12.76 µg/mL. The lowest value was observed against C. albicans, for which cell viability decreased significantly. SEM analysis of the biofilm formed by C. albicans in the absence of rottlerin revealed the presence of hyphae and pseudohyphae, important structures linked to microbial virulence, and many microbial aggregates. This was not observed in the biofilm of this same yeast formed in the presence of rottlerin. As for the biofilm formed by C. auris in the absence of rottlerin, a dense layer of adhered cells emerged, with the oval morphology characteristic of yeasts. In the presence of rottlerin, the amount of microbial aggregates decreased, and the yeasts became flattened. In the biofilm formed by C. dubliniensis (MYA-646), there was strong cell adhesion, with clear identification of the polymeric extracellular substance joining one cell to another. In the presence of rottlerin, the matrix decreased and the yeast cells detached. In addition, the cell wall was damaged, and holes appeared in the central region of the cell. The results presented here are unprecedented and promising: in vitro, rottlerin was able to inhibit both biomass and cell viability of the biofilm produced by Candida species, including C. auris, an emerging yeast that has been causing many infections in Brazil and worldwide. Palavras-chave: antibiofilm activity, Candida species, rottlerin, Scanning Electron Microscopy (SEM) Agência de fomento:Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) e Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). | |||||