Anidulafungin

In vitro susceptibility testing was performed using broth microdilution for filamentous fungi according to EUCAST guidelines with concentrations ranging from 0.016 µg/mL to 8 µg/mL. The antifungals used were amphotericin B (AmB), voriconazole (VRC), itraconazole (ITC), posaconazole (POS), and anidulafungin (ANI), all purchased from Sigma-Aldrich, reconstituted in DMSO and stored frozen at −80 °C until use. The MICs were determined in flat-bottomed 96-well plates with conidial suspensions prepared in RPMI 1640 supplemented with 2% glucose, buffered with MOPS, and adjusted to a final concentration of 5 × 10⁵ CFU/mL, as previously described [39 ]. Inoculated plates were incubated for 72 h at 35 °C. MICs were spectroscopically determined at 405 nm using a spectrophotometer (NEPHELOstar, BMG Labtech) as the antifungal concentration that resulted in 90% (AmB) or 50% growth inhibition (VRC, ITC, POS, ISA).

Caspofungin from two different sources was used to verify the consistency of our reported phenomena: caspofungin diacetate from Sigma-Aldrich (St. Louis, MO, USA) and caspofungin acetate from Merck (Rashway, NJ, USA). Micafungin sodium was provided by Astellas Pharma Inc. (Chuo-ku, Tokyo, Japan). Anidulafungin and amphotericin B were purchased from Sigma-Aldrich. Caspofungin and micafungin was dissolved in distilled water, amphotericin B and Anidulafungin was dissolved in 100% DMSO. Glucose [d-(+)-glucose] was purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). Three types of dH₂O were used in all experiments in order to verify the consistency of our reported phenomena: Invitrogen UltraPure DNase/RNase-free dH₂O, Otsuka dH₂O for 100% injection, and tap water purified using a Yamato Scientific Auto Still water purifier (WG250 with a 0.22 μm Sartolab RF filter; Sartorius, Göttingen, Germany).

POS, VRC, FLC, ITC, AMB, Anidulafungin (ANA), Micafungin (MFG), Nile Red, 2-deoxy-D-glucose (2-DOG), OME, Rabeprazole sodium (RAB), and Oligomycin (OM) were purchased from Sigma-Aldrich (St. Louis, MO, USA). OSP, CLO, Enniatin B (ENB), and Beauvericin (BEA) were purchased from Cayman Chemical (Ann Arbor, MI, USA). PON was purchased from Sellecheck Chemicals LLC (Houston, TX, USA) and VT-1161 was synthesized by MicroCombiChem (Wiesbaden, Germany). ISA was purchased from BOC Sciences (Shirley, NY, USA). The CLO analogs M1-M25 were designed, synthesized [73 (link),74 (link)], purified, and their structures confirmed using nuclear magnetic resonance (NMR), infrared spectroscopy (IR), and mass spectrometry (MS) (Supplemental Materials). The prodrugs OME and RAB were activated at low pH prior to the assay. OME was activated in SD-medium pH 3.5 for ten minutes. The assay was carried out at same pH as the active form of the compound is unstable at higher pH [75 (link)]. RAB is stable at higher pH and was activated for 1 h in the presence of 0.1 M HCl before diluting it into SD-medium with final pH 6.8 [76 (link)].

In vitro antifungal susceptibility testing of the 111 isolates was performed according to the EUCAST definitive document (E.DEF 9.3.1). Olorofim was provided by F2G, Ltd. (Manchester, UK). Comparator antifungal agents, including amphotericin B, itraconazole, voriconazole, posaconazole, isavuconazole, anidulafungin, caspofungin, and micafungin, were purchased from Sigma-Aldrich (MO, USA). The testing ranges for olorofim, voriconazole, and micafungin were 0.008 to 8 mg/liter, 0.002 to 0.2 mg/liter, and 0.004 to 0.4 mg/liter, respectively. The ranges for amphotericin B, itraconazole, posaconazole, isavuconazole, anidulafungin, and caspofungin were 0.016 to 16 mg/liter. For olorofim, endpoints were determined after 48 h of incubation at 100% inhibition compared with the growth control.
Resistant isolates were defined according to the EUCAST breakpoints (version 10.0). There are no clinical breakpoints available for echinocandins and olorofim. Candida parapsilosis ATCC 22019 and C. krusei ATCC 6258 were used as the quality control strains.

Amphotericin B, anidulafungin, micafungin, caspofungin, fluconazole and voriconazole were purchased from Sigma-Aldrich (Madrid, Spain). PTS and PTSO are organosulfur compounds present in onion extracts (AlioCare^TM) which were supplied with high purity (97%) by Enzim-Orbita Agroalimentares LDA (Tavira, Portugal) and dissolved in polysorbate-80 to a final concentration of 500,000 mg/L.

AFST was performed following the Clinical and Laboratory Standards Institute (CLSI) broth microdilution approach (CLSI-M27), which included fluconazole, micafungin, anidulafungin, and amphotericin B (AMB) (all sourced from Sigma, St. Louis, MO, USA). The 96-well plates containing the drug and the isolated suspensions were incubated for 24 hours at 37°C, after which the minimum inhibitory concentrations (MICs) were determined visually. Isolates with MIC values ≥8 µg/mL for fluconazole and echinocandins were denoted as fluconazole and echinocandin resistant, respectively. The MIC values for AMB were reported as wildtype (WT < 2 µg/mL) or non-WT (>2 µg/mL) given the lack of defined clinical breakpoints. C. parapsilosis (ATCC 22019) and Pichia kudriavzevii (ATCC 6258) type strains were included for quality control purposes.