Flucytosine

The in vitro susceptibility tests by using the CLSI reference broth microdilution method were performed for all species isolates number less than 100 strains. C. albicans and C. glabrata were randomly selected for the test. Those include 1272 C. albicans strains (including 998 isolates from VVC and 274 from RVVC) and 267 C. glabrata strains(including 197 isolates from VVC and 70 from RVVC). The MIC of Candida for all agents was read following 24–48 h incubation. The antifungals used were amphotericin B (Sigma, USA), Anidulafungin(Selleckchem, USA), Butoconazole(Sigma, USA), Caspofungin(Sigma, USA), Clotrimazole(Sigma, USA), Fluconazole(Sigma, USA), Flucytosine(Sigma, USA), Itraconazole(Sigma, USA), Micafungin(Selleckchem),Miconazole(Sigma, USA), Nystatin (Amresco, USA), Terbinafine(Santa Cruz, USA),Terconazole(Sigma),and Voriconazole(Fluka, USA). Quality control was performed as recommended in CLSI documents M27-A3 and M60 by using ATCC 90028 which is a reference strain of C. albicans and all results of the control were within established ranges [20 , 21 ].

Test compounds were diluted in sterile 96-well microplates with the redox-sensitive dye resazurin [49 (link)]. For this purpose, 10 μL of the test extract concentration was added to each well together with 190 μL of bacteria inoculum (5 × 10⁵ CFU/mL) or yeast inoculum (5 × 10³ CFU/mL). Incubation time varied depending on the microorganism from 7 days at 27 °C (mold), 24 h at 37 °C (yeast), and 17 h at 37 °C (bacteria). After this time, 10 μL of resazurin at 50 μg/mL per well was added. After this incubation period (under the same temperature condition previously declared) and times ranging from two and one days for, respectively, A. fumigatus and C. albicans, and 15 and 45 min for, respectively, S. aureus and E. coli. Corresponding fluorescence signals were measured at λ_ex = 550 nm and λ_em = 590 nm in a microplate reader (TECAN GENios, Germany). Microbial growth after treatment was compared to untreated-control wells (100% cell growth) and medium-control wells (0% cell growth). Doxycycline, flucytosine, and terbinafine (all purchased from Sigma-Aldrich, St. Louis, MO, USA) were used as a reference drugs. Mean IC₅₀ values estimated using linear regression analysis are presented with the SD of replicates (n = 2).

The Clinical and Laboratory Standards Institute document M27-A4 (CLSI M27-A4) broth microdilution protocol was used as a guideline for broth dilution method as an
assay for testing antifungal susceptibility of yeasts [ 19
]. Susceptibility of the isolates to fluconazole (Sigma Chemical Co, St Louis, Mo), amphotericin B (Bristol-Myers SP, Dublin, Ireland), flucytosine (Sigma Chemical Co),
itraconazole (Janssen-Cilag, High Wycombe, UK), voriconazole (Pfizer Inc., New York, NY), and caspofungin (Merck Sharp &Dohme, Whitehouse Station, NJ) were tested.
Stock and working concentrations of these drugs were prepared in 96-well microtiter plates based on the instructions stated by CLSI M27-A4 [ 19
]. A suspension equivalent to 0.5 McFarland was prepared from an overnight yeast culture on SDA by suspending the fungal cells in 10 mL of sterile distilled water,
and the concentration was spectrophotometrically adjusted according to CLSI M27-A4. Subsequently, 100 µL of fungal inocula were dispensed into wells of microplates,
and results were read after 24 h of incubation at 35 °C. Two quality control (QC) strains, C. parapsilosis ATCC 22019 and C. krusei ATCC 6258 were also included.
In the current study, interpretation of results was based on the breakpoints established for Candida species in CLSI- document M27-A4 (19).