Checkerboard assay

To determine the susceptibility of the selected strains to these drugs and to observe the effects of the drug combinations on them, we used CLSI M27-A3 checkerboard microdilution assay (CLSI, 2008). Briefly, drug dilutions and combinations were prepared in RPMI medium in microtiter plates. Negative (only medium) and positive (strain and medium without drug) controls were included. Strains were grown on Sabouraud agar at 35 °C and, after 24 h, one colony was transferred in 1 ml 0.85% saline solution. Inoculum was prepared by diluting yeast cells in RPMI medium to 3–5 × 10³ cells/ml using the automatic ImageJ-counting technique (Zupan et al., 2013). A total of 100 µl of this cell suspension was then transferred to 100 µl of drug suspension prepared in microplates. When the DMSO was used for drug dilution, it comprised <1% of the final test volume in the microtiter well. Tested drug concentrations ranged from 200–2 mg/l for MTX, 400–6.25 mg/l for AMX, 120–2 mg/l for MPA, 400–0.25 mg/l for Fk506, 16–0.125 mg/l for CsA, 256–0.25 mg/l for FLC, 256–0.25 mg/l for ITC, 1–0.004 mg/l for AMB, 16–0.068 mg/l for KCT, 16–0.017 mg/l for VRC, and 16–0.25 mg/l for POS. After incubation at 37 °C for 24, 48, or 72 h, we measured the optical density at 600 nm (OD₆₀₀) with a microplate reader (Tecan, Männedorf, Switzerland). Background optical densities were subtracted from that of each well. In vitro susceptibility and drug combination tests were performed at least in biological triplicates.

When we further investigated the observed mechanism of the antagonism between MPA and additional azole antifungals versus C. glabrata ATCC 2001 and Cgpdr1Δ mutant, we used a version of the assay described above but replaced SAB and RPMI media with YPD agar plates and broth, respectively. We have confirmed that the antagonistic effect in C. glabrata ATCC 2001 is present in both RPMI and YPD media, results are in Supplemental Information 1.