Biogenic nanosilver synthesized in Metarhizium robertsii waste mycelium extract - As a modulator of Candida albicans morphogenesis, membrane lipidome and biofilm.

Due to low efficacy of classic antimicrobial drugs, finding new active preparations attracts much attention. In this study an innovative, cost-effective and environmentally friendly method was applied to produce silver nanoparticles (AgNPs) using filamentous fungi Metarhizium robertsii biomass waste. It was shown that these NPs possess prominent antifungal effects against C. albicans, C. glabrata and C. parapsilosis reference strains. Further detailed studies were performed on C. albicans ATCC 90028. AgNPs kill curve (CFU method and esterase-mediated reduction of fluorescein diacetate); fractionally inhibitory concentration index (FICI) with fluconazole (FLC); effect on fungal cell membrane permeability (propidium iodide (PI) staining), membrane lipids profile (HPLC-MS), yeast morphotypes and intracellular reactive oxygen species level (H2DCFDA probe) were investigated. Anti-adhesive and anti-biofilm properties of AgNPs (alone and in combination with FLC) were also tested. Biosafety of AgNPs use was assessed in vitro in cytotoxicity tests against L929 fibroblasts, pulmonary epithelial A549 cell line, and red blood cells. Significant reduction in the viability of yeast cells treated with AgNPs was shown within 6 h. The proportion of C. albicans PI-positive cells increased in a dose and time-dependent manner. Changes in the qualitative and quantitative profile of cell membrane lipids, including significant decline in the quantity of most phospholipid species containing C18:2 and an increase in the amount of phospholipids containing C18:1 acyl species were observed after yeast exposure to AgNPs. CLSM images showed an enhancement in ROS intracellular accumulation in C. albicans treated with biogenic nanosilver. C. albicans transformation from yeast to hyphal forms was also reduced. AgNPs decreased adhesion of yeast to abiotic surfaces, as well as acted synergistically with FLC against sessile population. At fungicidal and fungistatic concentrations, they were non-toxic to mammalian cells. Obtained results confirm suitability of our "green synthesis" method to produce AgNPs with therapeutic potential against fungal infections.