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Molecules 2020, 25, 2023 10 of 14 form biofilms, a complex of cells, DNA, polymeric matrix, etc., on the surface of biomaterials and on catheters and prosthetic devices. The polymeric matrix has a crucial role in the antifungal resistance since it prevents the antimicrobial penetration inside the biofilm, allowing the internal cells to survive. Due of their intrinsic resistance to almost all antifungals in clinical use, increased resistance to this therapy, and the ability of cells within biofilms to withstand host immune defenses, the antifungal resistance of biofilms results most probably from the conjunction of several mechanisms that act in a time-dependent manner. Previous reports showed the antibiofilm activity of nanoparticles depends on their physicochemical properties, such as the size [28]. Similar to previously reported research by these authors, PchNPs of ca. 45 nm were able to eradicate biofilm of C. albicans. From these results, the biogenic silver nanoparticles synthesized in this work would be a useful tool for the development of biofilm disruption materials. 3. Materials and Methods 3.1. Synthesis of Silver Nanoparticles Strain of Phanerochaete chrysosporium (12G) from the Cátedra de Microbiología General Collection CCMG, Facultad de Química (Montevideo, Uruguay), was used for the nanoparticle biosynthesis. The mycelia were grown in Potato Dextrose Agar (PDA, BD Difco, Sparks, MD, USA) at 28 ◦C and two plugs of 0.9 cm in diameter were then transferred to 500 mL flasks containing 100 mL Potato Dextrose Broth (PDB, BD Difco). Fermentation was carried out at 28 ◦C, with agitation on an orbital shaker operating at 150 rpm for 72 h. The biomass from cultures was harvested by filtration and then washed extensively with sterilized distilled water to remove any remaining media components. Then, synthesis of silver nanoparticles was carried out as described in Sanguiñedo et al. [7]. Wet fungal mycelia were suspended in sterilized distilled water (0.1 g/mL) and incubated with agitation on an orbital shaker operating at 150 rpm. Then, the cell-free filtrate was collected by filtration of this suspension through membrane filter with 0.45 μm pore size. Finally, 50 mL of the cell-free filtrate was added to 50 mL of a silver nitrate solution. The mixture was incubated in dark. The absorbance spectrum was measured in the range of 250–800 nm and the maximum peak was determined, at different times. The reaction was stopped when there was no increase in the maximum absorption peak of silver nanoparticles. The remaining cell-free filtrate was used as control. Percentage yield was calculated according to weight of lyophilized silver nanoparticles and weight of silver nitrate used in the reaction, as previously reported [26]. To evaluate the incidence of the reaction variables in the biosynthesis of the nanoparticles, the following experimental conditions were modified: incubation time of the mycelia with water, concentration of AgNO3 and incubation temperature in the synthesis reaction. After the synthesis reactions, the samples were centrifuged at 10,000 rpm for 10 min. The supernatant was removed and nanoparticles (PchNPs) were washed twice using sterilized distilled water, by centrifuging the nanoparticles for 10 min at 10,000 rpm. The absorbance peak of the purified silver nanoparticles was measured and the concentration was estimated according to Paramelle et al. [29]. 3.2. Characterization of Silver Nanoparticles 3.2.1. UV-Vis Spectroscopy The absorbance spectrum was measured in the range of 250–800 nm, at predetermined time intervals. Also, the color changes of reaction mixtures were used as evidence for silver nanoparticles formation.PDF Image | Biofilm Eradication Using Biogenic Silver Nanoparticles
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