Susceptibility testing.

EUCAST MICs were determined following E.Def 7.3.1 methodology (12). Manogepix (APX001A; Amplyx Pharmaceuticals, San Diego, USA) pure substance was stored in aliquots at −80°C, and stock solutions were prepared in dimethyl sulfoxide (DMSO) (Sigma-Aldrich, Brøndby, Denmark; 5000 mg/liter). The final drug concentration ranges studied were 0.001 to 0.5 mg/liter. The following comparator compounds were also investigated (source of compound and final concentration range in parentheses): anidulafungin (Pfizer A/S, Ballerup, Denmark; 0.004 to 4 mg/liter), micafungin (Astellas Pharma Inc., Tokyo, Japan; 0.004 to 4 mg/liter), amphotericin B (Sigma-Aldrich; 0.004 to 4 mg/liter), fluconazole (Sigma-Aldrich; 0.03 to 32 mg/liter), and voriconazole (Pfizer A/S, Ballerup, Denmark; 0.004 to 4 mg/liter). Microtiter plates with 2-fold dilutions prepared in double-concentrated medium according to the EUCAST methodology were prepared using serial dilution and pipette tip changes for every fourth well. Cell culture-treated (Nunc MicroWell 96-well microplates; Thermo Fisher Scientific; catalog no. 167008) were used throughout and frozen at −80°C prior to use. The EUCAST quality control (QC) strains C. albicans CNM-CL-F8555, C. parapsilosis ATCC 22019, and C. krusei ATCC 6258 were tested in parallel. MICs for the licensed compounds were classified as wild type and non-wild type, adopting the EUCAST epidemiological cutoff values (ECOFFs) valid 4 February 2020 (https://www.eucast.org).