2.8. Activity of ABC Efflux Pumps on Conidial- and Biofilm-Forming Cells

Both conidial and 72 h-old-biofilm-forming cells (1 × 10⁷/mL, initial inoculums) were incubated with 10 µM rhodamine 6G (R6G, Sigma-Aldrich) for 1 h at 37 °C, as described by Silva et al. [57]. Then, the supernatants were discarded, and the systems were washed with PBS and incubated for additional 1 h at 37 °C in PBS containing 2% glucose. The supernatants were then collected, and fluorescence was measured on a fluorimeter (excitation, 529 nm; emission, 553 nm). R6G non-stained fungal cells were used as a control. In addition, F. pedrosoi and P. verrucosa were treated with MIC (both 0.19 µM) determined for planktonic cells and with the highest concentration (800 µM) tested before for biofilm-forming cells. The efflux pump inhibitor phenylalanine-arginine beta-naphthylamide (PAβN, Sigma-Aldrich) was used to evaluate the possible role of efflux pumps in antifungal resistance. The bMIC for itraconazole in the presence of PAβN (64 µg/mL) was determined after 48 h of incubation according to CLSI [56], with modifications.