Isolates detected as resistant by the screening method were further tested for antifungal susceptibility using the European Committee for Antimicrobial Susceptibility Testing (EUCAST) microdilution broth reference method [30 ]. All clinically available azole antifungal molecules were tested: itraconazole (ITZ) (Sigma-Aldrich); voriconazole (VCZ) (Sigma-Aldrich); posaconazole (PCZ) (MSD, Kenilworth, NJ, USA); and isavuconazole (ISA) (Basilea Pharmaceutica International Ltd., Basel, Switzerland). The number of conidia inoculated was 105 to 2.5 × 105 cfu/mL and the concentrations of each drug ranged from 0.016 to 8 mg/L. After 48 h of incubation at 37 °C, the minimum inhibitory concentration (MIC) was determined, both visually, as the lowest drug concentration that could completely inhibit fungal growth, and spectrophotometrically at 550 nm using a 90% growth inhibition endpoint. All tests were performed in triplicate. Candida parapsilosis ATCC 22019 and Candida krusei ATCC 6258 were included as quality controls.
Voriconazole vcz
Manufactured by Merck Group
Sourced in United States
Voriconazole (VCZ) is a pharmaceutical compound used as an antifungal agent. It is a triazole derivative that functions as an inhibitor of fungal cytochrome P450-dependent 14α-sterol demethylase, which is an essential enzyme in the biosynthesis of ergosterol, a key component of the fungal cell membrane.
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4 protocols using voriconazole vcz
1
Antifungal Susceptibility Testing Protocol
2
Antifungal Susceptibility Determination
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For antifungal susceptibility determination, the MICs of TMMI-012 and S. boydii CBS 120157 were determined according to recommendations stated in the Clinical and Laboratory Standards Institute (CLSI) M38-A2 document [17 (link)]. According to the CLSI method, antifungal drugs used, including amphotericin B (AMB), itraconazole (ICZ), posaconazole (PSZ), voriconazole (VCZ) (Sigma, St. Louis, MO, USA) were obtained as reagent-grade powders. TMMI-012 and S. boydii CBS 120157 inocula were diluted in Gibco Roswell Park Memorial Institute (RPMI) 1640 medium, and the final inoculum in assay wells was between 0.5 × 103–5 × 103 CFU/mL and incubated at 35 °C for 24–48 h. According to the CLSI method, the MICs were visually determined after 24 h of incubation by selecting the lowest concentration of a drug that caused ≥50% inhibition in growth compared to drug-free controls.
3
In vitro Antifungal Compound Assays
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For the in vitro assays, voriconazole (VCZ, Sigma-Aldrich), itraconazole (ITZ, Sigma-Aldrich), amphotericin B (AMB, Sigma-Aldrich), terbinafine (TRB, Sigma-Aldrich), and brilacidin (BRI, supplied by Innovation Pharmaceuticals) were diluted in dimethyl sulfoxide (DMOS); while miltefosine (MFS, Sigma-Aldrich), the milteforan active compound, was diluted in ethanol; and caspofungin (CSP, Sigma-Aldrich) was diluted in distilled water. milteforan (miltefosine 2%) was purchased from Virbac as an oral solution.
4
Antifungal Activity of Peptides Against Candida albicans
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Candida albicans (ATCC© 10231TM) was cultured in Sabouraud medium (Sigma-Aldrich) supplemented with tetracycline (10 μg/mL) and cefotaxime (10 μg/mL) at 37 °C for 24 h. Candida albicans (OD600nm = 0,001) were plated in 96-well plates and treated either with different concentrations of the peptides of interest, and/or with different concentrations of voriconazole (VCZ) (Sigma-Aldrich). As a positive control, cells were treated with 10 μg/mL VCZ. After 24 hours incubation, yeast growth was assessed by optical density OD600nm using a spectrophotometer (Multiscan EX).
The MIC of the antifungal agents, defined as the lowest concentration of drug able to inhibit 100% of the growth of a pathogen was determined using a modified Gompertz model 47 (link).
The efficiency of the combination between D-Ctl and VCZ was determined by the calculation of the Fractional inhibitory concentration index (FICI) according to the following formula: FICI = FICD-Ctl + FICVCZ = (MICD-Ctl in combination/MICD-Ctl + (MICVCZ in combination/MICVCZ . According to EUCAST38 , the effect of the combination is synergistic if FICI ≤ 0,5, additive if 0,5 < FICI ≤ 1, indifferent if 1 < FICI < 2 and antagonistic if FICI ≥ 2.
The MIC of the antifungal agents, defined as the lowest concentration of drug able to inhibit 100% of the growth of a pathogen was determined using a modified Gompertz model 47 (link).
The efficiency of the combination between D-Ctl and VCZ was determined by the calculation of the Fractional inhibitory concentration index (FICI) according to the following formula: FICI = FICD-Ctl + FICVCZ = (MICD-Ctl in combination/MICD-Ctl + (MICVCZ in combination/MICVCZ . According to EUCAST38 , the effect of the combination is synergistic if FICI ≤ 0,5, additive if 0,5 < FICI ≤ 1, indifferent if 1 < FICI < 2 and antagonistic if FICI ≥ 2.
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