Phenotypic Analyses

Strains were recovered from our glycerol stock collection and grown for 2 days at 37°C on YPD agar. Single colonies were cultivated in 15 mL YPD broth (37°C, 200 rpm, overnight). Then, each sample was diluted to an optical density (OD) at 600 nm of 0.2 in 3 mL of YPD broth and grown for 3 h more in the same conditions (37°C, 200 rpm). Dilutions were made again to have an OD at 600 nm of 0.5 in 1 mL of YPD broth in order to have the same amount of cell in all the experiments. The samples were centrifuged for 2 min at 3000 ×g, washed with 1 ml of sterile water and centrifuged again for 2 min more at 3000 ×g for a final resuspension of the pellet in 1 ml of sterile water. At the end, 5 μL of each sample was inoculated in 95 μL of the corresponding medium in a 96-well plate format. All experiments were run in triplicate.

Six different growth conditions were tested: oxidative stress was assessed by measuring the growth of the cultures on YPD broth supplemented with 10 mM H₂O₂, reductive stress with 2.5 mM DTT and osmotic stress with 1 M NaCl; high temperature (41.5°C), pH = 2 and pH = 9 along with the control growth in standard YPD at 37°C. Cultures were grown in 96-well plates at 37 or 41.5°C, shaking, for 20 or 72 h depending on the growth rate in each condition, and monitored to determine the optical density at 600 nm every 10 min by a TECAN Infinite^® M200 microplate reader. Growthcurver v0.2.1, an R package, was used to measure growth (Sprouffske and Wagner, 2016).

Biofilm formation was assessed as described previously (Gómez-Molero et al., 2015). Studied isolates and controls (CBS138, moderate biofilm formation capacity; PEU-382 and PEU-427, high biofilm formation capacity) were cultured overnight in YPD medium at 37°C. The optical density was determined at 600 nm (Ultrospec 1000) and adjusted to a value of 2 using sterile NaCl 0.9%. Fifty microliters aliquots of the cell suspensions were placed into 96-well polystyrol microtiter plates (Greiner Bio-One) and incubated for 24 h at 37°C. The medium was removed and the attached biofilms washed once with 200 μL distilled water. Cells were stained for 30 min in 100 μL of 0.1% (w/v) crystal violet (CV) solution. Excess CV was removed and the biofilm carefully washed once with 200 μL distilled water. To release CV from the cells, 200 μL 1% (w/v) SDS in 50% (v/v) ethanol was added and the cellular material resuspended by pipetting. CV absorbance was quantified at 490 nm using a microtiter plate reader (MRX TC Revelation). Final data is the average of the three independent biological experiments, each one with four technical repeats.

Isolates were cultured overnight on YPD agar plates. After that, antifungal drug susceptibilities toward Fluconazole, Isavuconazole, Posaconazole, Voriconazole, Micafungin, Caspofungin, 5-Fluorocytosine, and Amphotericin B were determined according to EUCAST EDef 7.1 method (Arendrup et al., 2012). The MIC values of each SAT strains were calculated according to EUCAST guidelines¹.