In vitro biofilm inhibition assay in microtiter plates

The biofilm inhibition assay in polystyrene microtiter plates was performed as described previously (Vriens et al., 2015b), except for the quantification method. Briefly, Candida cells were incubated at OD_{600 nm} = 0.1 in RPMI in round-bottomed microtiter plates (TPP, Tradingen, Switzerland). After 1 h of adhesion at 37°C, biofilms were washed and subsequently incubated for 24 h with fresh RPMI containing HsLin and/or an echinocandin (Vriens et al., 2015b). All C. albicans biofilms were quantified by incubating the treated biofilms for 1 h at 37°C with 1/100 dilution of the metabolic dye CTB (Promega, Madison, USA) in PBS (Vriens et al., 2015b), except for the C. albicans and non-albicans Candida species biofilms in Figure 5, which were quantified using XTT. In the latter, 110 μL of a XTT solution (0.25 mg/mL in PBS, 1 μM menadione; Sigma Aldrich, St Louis, USA) was added to the biofilms. After 1 h at 37°C, the absorption (OD_{490 nm}) of 100 μL of the converted XTT solution was measured. Next, the Biofilm Inhibitory Concentration 50 (BIC₅₀) value, i.e., the concentration of the compound required for 50% biofilm formation inhibition, compared to the control treatment (0.5% DSMO), was determined. Checkerboard assays were performed for [echinocandin + HsLin] combinations (Vriens et al., 2015b), and the fold change of echinocandin's BIC₅₀ was determined, i.e., the ratio of echinocandin's BIC₅₀ in the absence/presence of the peptide. To test synergistic interactions between two agents against biofilm formation, the Fractional Inhibitory Concentration Index (FICI) was calculated (Odds, 2003; Delattin et al., 2014).