Fluconazole flu

The evaluation of the antifungal activity by determining the MIC was performed by the microplate dilution technique according to the protocol described in the M27-A3 document [35 ] with modifications. The concentration of the EO and citronellal was 7.8 to 1000 μg/mL. The EO was dissolved in 10% methanol and 2% Tween 80. A quantity of 0.1 mL was placed in a 96-well microtiter plate containing Roswell Park Memorial Institute (RPMI) 1640 medium. Each well was inoculated with 0.1 mL of a suspension containing 2.5 × 10³ CFU/mL of yeast.
Amphotericin B (AmB) (Sigma-Aldrich^®) and fluconazole (FLU) (Sigma-Aldrich^®) were used as the positive controls. Additional controls also included the culture medium, yeast growth, EO, and solvent. The microplates were incubated at 37 °C for 48 h. After incubation, 20 μL of an aqueous 2% solution of 2,3,5-triphenyltetrazolium chloride (TTC) was added, the plates were incubated at 37 °C for 2 h [32 (link)], and absorbance of the samples was measured by spectrophotometer (Biospectro, SP22, Curitiba, Brazil). All tests were performed in triplicate.
According to obtained results of the EO MIC determination, the more sensitive strains (one ATCC and one clinical strain of each species) were selected to evaluate the antifungal activity of citronellal.

A total of 31 L. elongisporus isolates recovered from clinical specimens (n = 13 BSIs) and inanimate-environment isolates (n = 2) from the NICU, from the floor of two other hospitals (n = 3), and from the surfaces of stored apples (n = 13) were subjected to antifungal susceptibility testing (AFST) by the CLSI broth microdilution method following method M27-A3 (15 ). The isolates were tested against 10 antifungal drugs of three classes, i.e., azoles, echinocandins, and amphotericin B, as detailed in Table 2. The antifungals tested were fluconazole (FLU; Sigma, St. Louis, MO, USA), itraconazole (ITC; Lee Pharma, Hyderabad, India), voriconazole (VRC; Pfizer, Groton, CT, USA), posaconazole (POS; Merck, Whitehouse Station, NJ, USA), isavuconazole (ISA; Basilea Pharmaceutical, Basel, Switzerland), 5-flucytosine (5-FC; Sigma), caspofungin (CFG; Sigma), micafungin (MFG; Sigma), anidulafungin (AFG; Sigma), and amphotericin B (AMB; Sigma). The drugs were tested for 10 (2-fold) dilutions, and the drug concentration ranges were as follows: FLU, 0.25 to 128 mg/L; ITC, VRC, and AMB, 0.03 to 16 mg/L; POS, ISA, AFG, MFG, and CFG, 0.015 to 8 mg/L; 5-FC, 0.125 to 64 mg/L. Candida krusei strain ATCC 6258 and Candida parapsilosis strain ATCC 22019 were used as quality control strains.

Stock solutions of two commercial antimicrobial agents, Fluconazole (FLU, Sigma-Aldrich, St. Louis, MO, USA) and Gentamicin sulfate (GEN, Sigma-Aldrich, St. Louis, MO, USA) were prepared and stored according to the manufacturer’s instructions. Also, two different honeys, Portuguese heather honey (raw dark amber honey whose main plant nectar is heather (30%, APISMaia company (Porto Portugal)) collected by a beekeeper in the North of Portugal) and manuka (commercial Medihoney^®) were stored at 4 °C, and the dilutions were prepared with RPMI 6420 medium. All the concentrations of different antimicrobial agents (natural and commercial) tested in this work are presented in Table 1.

The broth microdilution method (as recommended by EUCAST [75 ]) was used to determine the MIC of arene-ruthenium(II) complexes with carbotioamidopyrazole derivatives 3a–3d, NH₄PF₆ and selected antibiotics: oxacillin (Oxa), gentamicin (Gen), and fluconazole (Flu) (all antibiotics were purchased from Sigma, St. Louis, MO, USA). Stock solutions of the lyophilized compounds were freshly prepared in 100% DMSO (POCh, Gliwice, Poland) for the complexes, or in water for injection (Sigma, St. Louis, MO, USA) in the case of antibiotics. Following this, two-fold dilutions were prepared in liquid culture medium to the following final concentration range: 31.2–1000 µg/mL in the first series (the complexes and NH₄PF₆ against all tested microorganisms), 1.95–250 µg/mL in the second series (the complexes against staphylococci), 0.03–2 µg/mL (Gen against P. vulgaris), and 0.25–16 µg/mL (Flu against C. albicans). MIC and MBC/MFC were defined as the lowest concentrations of the compounds inhibiting bacterial/fungal growth or able to kill added microbial inoculum, respectively, and were evaluated as described by Namiecinska et al. [17 (link)]. Experiments were carried out in duplicate.