Micafungin

Antifungal susceptibility was determined in flat bottom, 96-well microtiter plates (Sarstedt) using a modified broth microdilution protocol, as described [21] (link). Minimum inhibitory concentration (MIC) tests were set up in a total volume of 0.2 ml/well with 2-fold dilutions of micafungin (MF, generously provided by Julia R. Köhler) or caspofungin (CS, generously provided by Rochelle Bagatell). Echinocandin gradients were typically from 2 µg/ml down to 0 with the following concentration steps in µg/ml: 1, 0.5, 0.25, 0.125, 0.0625, 0.03125, 0.015625, 0.0078125, 0.00390625, 0.00195313. For gradients from 16 µg/ml down to 0, the concentration steps in µg/ml were: 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.0625, 0.03125, 0.015625. Cell densities of overnight cultures were determined and dilutions were prepared such that ∼10³ cells were inoculated into each well. Geldanamycin (GdA, A.G. Scientific, Inc.) and radicicol (RAD, A.G. Scientific, Inc.) were used to inhibit Hsp90 at the indicated concentrations, and cyclosporin A (CsA, CalBiochem) and FK506 (A.G. Scientific, Inc.) were used to inhibit calcineurin at the indicated concentrations. Checkerboard assays were set up in a total volume of 0.2 ml/well with 2-fold dilutions of MF across the x-axis of the plate and 2-fold dilutions of either GdA or CsA across the y-axis of the plate. Plates were inoculated as with MIC tests. Dimethyl sulfoxide (DMSO, Sigma Aldrich Co.) was the vehicle for GdA, RAD, CsA, and FK506. Sterile water was the vehicle for MF and CS. Plates were incubated in the dark at 30°C for the time period indicated, at which point plates were sealed and re-suspended by agitation. Absorbance was determined at 600 nm using a spectrophotometer (Molecular Devices) and was corrected for background from the corresponding medium. Each strain was tested in duplicate on at least two occasions. MIC data was quantitatively displayed with color using the program Java TreeView 1.1.3 (http://jtreeview.sourceforge.net).

Antifungal tolerance and resistance were determined in flat bottom, 96-well microtiter plates (Sarstedt) using a modified broth microdilution protocol as described [25] (link), [27] (link). Dimethyl sulfoxide (DMSO, Sigma Aldrich Co.) was the solvent for fenpropimorph (FN, Sigma Aldrich Co) and terbinafine (TB, Sigma Aldrich Co.); fluconazole (FL, Sequoia Research Products) and micafungin (MF, generously provided by Julia R. Köhler) were dissolved in sterile ddH₂O. Geldanamycin (GdA, A.G. Scientific, Inc.) was used to inhibit Hsp90 at the indicated concentrations. Cyclosporin A (CsA, Calbiochem) was used to inhibit calcineurin at the indicated concentrations. Cercosporamide and staurosporine (STS, A.G. Scientific, Inc.) were used to inhibit protein kinase C at the indicated concentrations. DMSO was the solvent for GdA, CsA, STS, and cercosporamide.
Minimum inhibitory concentration (MIC) tests were set up in a total volume of 0.2 ml/well with 2-fold dilutions of FL, FN, TB and cercosporamide. FL gradients were from 256 µg/ml down to 0 with the following concentration steps in µg/ml: 256, 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25. FN gradients were from 25 µg/ml down to 0 with the following concentration steps in µg/ml: 25, 12.5, 6.25, 3.125, 1.5625, 0.78125, 0.390625, 0.1953125, 0.09765625, 0.04882813, 0.02441406. TB gradients were from 250 µg/ml with the following concentration steps in µg/ml: 250, 125, 62.5, 31.25, 15.625, 7.8125, 3.90625, 1.953125, 0.9765625, 0.48828125, 0.24414063. Cercosporamide gradients were from 100 µg/ml with the following concentration steps in µg/ml: 100, 50, 25, 12.5, 6.25, 3.125, 1.5625, 0.78125, 0.390625, 0.1953125, 0.09765625. Cell densities of overnight cultures were determined and dilutions were prepared such that ∼10³ cells were inoculated into each well. Plates were incubated in the dark at 30°C or 35°C for the period of time indicated in the figure legend, at which point plates were sealed with tape and re-suspended by agitation. Absorbance was determined at 600 nm using a spectrophotometer (Molecular Devices) and corrected for background from the corresponding medium. Each strain was tested in duplicate on at least 3 occasions. MIC data was quantitatively displayed with color using the program Java TreeView 1.1.1 (http://jtreeview.sourceforge.net).
Checkerboard assays were set up in a total volume of 0.2 ml/well with 2-fold dilutions of cyclosporin A across the x-axis of the plate and 2-fold dilutions of STS across the y-axis of the plate. STS gradients were from 0.5 µg/ml to 0 in the following concentrations steps in µg/ml: 0.5, 0.25, 0.125, 0.0625, 0.03125, 0.015625, 0.0078125. CsA gradients were from 48 µg/ml down to 0 in the following concentration steps in µM: 48, 24, 12, 6, 3, 1.5, 0.75, 0.375, 0.1875, 0.09375, 0.046875. Plates were inoculated and growth was measured as with MIC tests. To test for synergy, the fractional inhibitory concentration (FIC) was calculated as follows: [(MIC₈₀ of drug A in combination)/(MIC₈₀ of drug A alone)] + [(MIC₈₀ of drug B in combination)/(MIC₈₀ of drug B alone)]. Values of ≤0.5 indicate synergy, those of >0.5 but <2 indicate no interaction and those ≥2 indicate antagonism.

Neat 500 µM micafungin solution was serially diluted in 50/50 acetonitrile (ACN)/water to create neat standard curves and quality control spiking solutions. 10 µL of neat spiking solutions were added to 90 µL of drug-free macrophage lysate to create standard and QC samples. 10 µL of control, standard, or study sample lysate were added to 100 µL of a 50:50 acetonitrile: methanol protein precipitation solvent mix containing 10 ng/mL of the internal standard verapamil to extract micafungin. Extracts were vortexed for 5 min and centrifuged at 3700 × g for 5 min. 75 µL of supernatant was transferred for LC-MS/MS analysis and diluted with 75 µL of Milli-Q deionized water. LC-MS/MS analysis was performed on a Sciex Applied Biosystems Qtrap 6500+ triple-quadrupole mass spectrometer coupled to a Shimadzu Nexera X2 UHPLC system to quantify each drug concentrations in each sample. Chromatography was performed on an Agilent SB-C8 (2.1 × 30 mm; particle size, 3.5 µm) using a reverse phase gradient. Milli-Q deionized water with 0.1% formic acid was used for the aqueous mobile phase and 0.1% formic acid in acetonitrile for the organic mobile phase. Multiple-reaction monitoring of parent/daughter transitions in electrospray positive-ionization mode was used to quantify all analytes. The MRM transitions of 455.40/165.20, 1270.40/1190.40, 823.5/791.6, 402.2/358.0, 473.2/431.2 were used for verapamil, micafungin, rifampicin, moxifloxacin, and clofazimine, respectively. Data processing was performed using Analyst software (version 1.6.3; Applied Biosystems Sciex).

PBS washed initial inoculum of C. glabrata cells were stained with 200 µg/ml of FITC (Millipore Sigma) in carbonate buffer (0.1 M Na₂CO₃, 0.15 M NaCl, pH = 9.3) and incubated at 37 °C, followed by 3 times PBS washing. The THP1 macrophages were infected with MOI of 10/1 and after 3-, 6-, 24-, and 48-h pst with micafungin (0.125 µg/ml), the C. glabrata cells were released from macrophages and counterstained with 50 µg/ml AF-647-ConA (Millipore Sigma) in PBS buffer+2% BSA. The pellets were washed 3 times with PBS and subjected to flow cytometry (BD Biosciences). Double-stained yeasts were mother cells, while yeast cells only stained with AF-647 ConA represented replicated cells.

ICG cells (CBS138) treated with micafungin (0.125 µg/ml) for 24 h were exposed to 1 ml of fresh RPMI containing either amphotericin B (2 µg/ml) or micafungin (0.125 µg/ml) and the survival rate was assessed 24 and 48 h later. The survival rates of treated ICG were normalized against the counterparts treated with drug-free RPMI and the data were presented by percentage.