Determinação da ação antifúngica de nanopartículas de óxido de zinco sobre Cryptococcus neoformans

Abstract

The accumulation of pigeon excreta in public buildings, monuments and homes; lead to several risks of contamination in water and food sources. Among the various microorganisms transmitted through their excrement, Cryptococcus neoformans is the main causative agent of fungal meningitis in the world; mainly in immunocompromised individuals. Usually acquired by inhalation of Cryptococcus neoformans. In recent decades, it has been discovered that nanomaterials have antimicrobial activity, particularly zinc oxide nanoparticles (ZnO-NPs), which can be used as good antibacterial agents, both for Gram-positive and Gram-negative bacteria. Thus, this work proposed to evaluate the antimicrobial action of zinc oxide nanoparticles (ZnO-NPs) on planktonic cells and biofilms of Cryptococcus neoformans isolated from human lesions and fruit (ATCC 28957 - 32045). The synthesis of zinc oxide nanoparticles was carried out by simple precipitation. The determination of the minimum inhibitory concentration (MIC) of ZnO-NPs in planktonic cells of Cryptococcus neoformans was carried out using the broth microdilution method, according to the method proposed by the Brazilian Committee on Antimicrobial Susceptibility Testing with modifications. Biofilms were formed by incubated at 35 °C for 24 - 48 hours in 96-well plates. The biofilms were treated with ZnO-NPs at concentrations of 5,000 µg/mL, 2500 µg/mL, and 1250 µg/mL. These concentrations were selected for this analysis based on the results obtained from the MIC. The resulting biofilms were analyzed by quantifying colony forming units (CFUs), total biomass and metabolic activity. Statistical analysis was carried out verifying the normality of the distribution of values; using Shapiro-Wilk available in the R program (version 3.2.3), and two-way analysis of variance (Two-way anova), available in the statistical program Graphpad Prism (Version 5.0). The ZnO-NPs showed antifungal activity against planktonic cells, with Cryptococcus neoformans MICs of 19.5 for ATCC 28957, and 312 µg/mL for ATCC 32045 respectively. All tested concentrations of ZNO-Ns generated significant reductions (p<0.05) in biofilm metabolism, with dose-dependent effects, compared to the control group. ZNO-NPs showed dose-dependent effects on the total biomass of biofilms; evaluated only at the highest concentration (5000 µg/mL), where the greatest reductions were observed. All tested concentrations of ZNO-NPs generated significant reductions (p<0.05) in the quantification of cultivable biofilm cells, with dose-dependent effects compared to the control group. Although there are not many studies on the antibiofilm activity of ZnO-NPs from Cryptococcus neoformans; It is known that this nanoparticle has broad antifungal activity in planktonic form, and in biofilms of other yeasts. Therefore, it reinforces that the use of ZnO-NPs against pathogens is possible and promising.