Morpholinepropanesulfonic acid mops

The fungal isolate used in this study was a Candida glabrata clinical isolate (University of California Davis Medical Center, UCDMC). The fungi were grown and maintained in Sabouraud dextrose agar and incubated at 30 °C for 24 h. The in vitro susceptibility of oceanalin B was determined by the broth microdilution method [20 (link)]. Briefly, 2-fold serial dilutions of oceanalin B were prepared in 96-well microtiter plates from stock solutions in an RPMI-1640 broth medium (Sigma, St. Louis, MO, USA) buffered to a final pH of 7.0 with 0.165 M morpholinepropanesulfonic acid (MOPS; Sigma, St. Louis, MO, USA) to a final volume of 100 μL. A stock solution of oceanalin B was prepared in dimethyl sulfoxide and amphotericin B (AMB) (Sigma, St. Louis, MO, USA) was prepared as a positive control. C. glabrata cells (5 × 10⁶/mL) in 100 μL suspension was added to the wells. The final concentrations tested were from 0.062 to 64 μg/mL and from 0.0078 to 8 μg/mL for amphotericin B. The experiment was performed in triplicates in each run of the experiments. Cell growth was determined by the OD at 600 nm using a Spectramax Plus 384 microplate reader (Molecular Devices, San Jose, CA, USA). The MIC end point was defined as the lowest concentration with complete (>90%) growth inhibition.

Amphotericin B, itraconazole, caspofungin and tigecycline were obtained from Bristol-Myers Squibb (New York, USA), Sigma Aldrich (St. Louis, MO, USA), Merck Sharp Dohme (Kenilworth, NJ, USA) and Wyeth Pharmaceuticals (Madison, NJ, USA), respectively, while fluconazole and anidulafungin were acquired from Pfizer (New York, USA). Stock solutions from dry powders were prepared according to the manufacturers’ recommendation and stored frozen at −80 °C for up to six months. Final concentrations of antimicrobials were prepared in Roswell Park Memorial Institute (RPMI, Sigma-Aldrich, St. Louis, MO, USA) medium supplemented with L-glutamine and buffered with morpholinepropanesulfonic acid (MOPS; Sigma-Aldrich, St. Louis, MO, USA), prior to use.

Stock solution of amphotericin B (AMB; Sigma Chemical Co., St. Louis, MO, United States) was prepared as recommended by the document M27-A3 (CLSI, 2008 ). Serial two-fold dilutions of AMB were performed in RPMI 1640 medium (Sigma-Aldrich, MO, United States), buffered to pH 7.0 with 0.165 M morpholinepropanesulfonic acid (MOPS; Sigma-Aldrich, MO, USA).

The ZnO nanoparticle was purchased from Sigma-Aldrich (Taufkirchen, Germany). Amphotericin B (AMB) was purchased from Beijing Mesochem Technology Co., Ltd. (Beijing, China). Polyethylene glycol (PEG, 40 KDa) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Tween-80 was purchased from Merck Schuchardt OHG (Hohenbrunn, Germany). The used media RPMI 1640 and morpholinepropanesulfonic acid (MOPS) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Sabouraud dextrose agar (SDA) was purchased from Oxoid (Hampshire, UK). Levels of the liver enzymes and kidney functions, aspartate transaminase (AST), alanine transaminase (ALT), and creatinine assay kits were purchased from Sigma-Aldrich (St. Louis, MO, USA). Blood urea nitrogen (BUN) colorimetric detection kit was ordered from Thermo Fisher Scientific (Waltham, MA, USA). All other chemicals and solvent were purchased from Sigma-Aldrich.

The MIC value of violacein and azole antibiotics were performed using the microdilution assay as described in M38-A2, Clinical and Laboratory Standards Institute (CLSI, 2008 ), with minor modifications. The medium used was RPMI 1640 with L-glutamine (Sigma-Aldrich, St. Louis, MO, USA) buffered to pH 7.0 with 0.165 M morpholine propanesulfonic acid (MOPS) (Sigma-Aldrich, USA), supplemented with 2% glucose. The T. rubrum cell suspension was prepared in a 0.85% saline solution. The suspension of conidia was then transferred to sterile test tubes where they remained for 30 min to separate the microconidia, which were lighter and therefore, present in the supernatant. After that the separated microconidia concentration was adjusted to obtain a final concentration ranging from 3 × 10³ to 6 × 10³ CFU/ml. Then these suspensions were diluted in RPMI 1640 (Sigma-Aldrich, St. Louis, MO, USA) and inoculated on 96-well plates that had been previously prepared with the test compounds diluted at concentrations from 0.5 to 1000 μg/ml. Positive (medium with inoculum) and negative (medium alone) controls were included in all experiments. The plates were incubated for 7 days at 28°C. The MIC reading was performed by spectrophotometry at 600 nm. The MIC was defined as the concentration of violacein/azoles that inhibit 90% of fungal growth.

Micafungin (Astellas Pharma, Madrid, Spain) was dissolved in dimethyl sulfoxide (DMSO), to obtain a stock solution of 5120 μg/ml. The dilutions were prepared in RPMI 1640 medium with L-glutamine, 0.2% glucose and without NaHCO₂ buffered to pH 7 with 0.165 M morpholinepropanesulfonic acid (MOPS) (Sigma-Aldrich, Madrid, Spain). Stock solutions were stored at – 80°C until use.

The following antifungals were used in this study: SCY-078 (Scynexis, Inc., Jersey City, NJ), caspofungin (CASP; Lanospharma Laboratories Co., Ltd., Hong Kong), voriconazole (VRC; Sigma-Aldrich), and fluconazole (FLC; Sigma-Aldrich). Stock solutions (10 mg/ml) of these antifungals were prepared in dimethyl sulfoxide (DMSO; Sigma-Aldrich). Working solutions were prepared in RPMI 1640 medium (Sigma-Aldrich) buffered to a pH of 7.0 with 0.165 M morpholine-propanesulfonic acid (MOPS; Sigma-Aldrich). The final concentration of DMSO was ≤1% (vol/vol) of the solution composition. Stock solutions were separated into unit-of-use portions and stored at −80°C until used.