Mueller hinton broth medium

All strains were subcultured on trypticase soy agar with 5% sheep blood (Becton, Dickinson and Company, New Jersey) at 35 °C for 3‒5 days. Antimicrobial susceptibility testing was performed per the recommendations in the CLSI M24A-2 at 30 °C⁸ . A nephelometer (VITEK DENSICHEK, bioMerieux, France) was used to standardize the inoculum density (0.5 McFarland standard). The MICs of 24 antimicrobial agents (tigecycline, linezolid, clarithromycin, azithromycin, arbekacin, amikacin, gentamycin, tobramycin, imipenem, doripenem, faropenem, levofloxacin, sitafloxacin, ciprofloxacin, moxifloxacin, cefmetazole, cefoxitin, ceftriaxone, cefepime, ethambutol, rifabutin, minocycline, amoxicillin/clavulanic acid, and trimethoprim/sulfamethoxazole) were measured by the micro-dilution method using cation-adjusted Mueller–Hinton broth medium (Becton, Dickinson and Company)⁸ . The MICs of clarithromycin and azithromycin were read two times to detect induced resistance. Positive growth of the control between days 3 and 5 was defined as early-reading-time. Inducible macrolide resistance was determined on day 14 and defined as LRT. Repetition of MIC measurement, as recommended by guidelines, was performed without exception.

Minimal Inhibitory Concentration (MIC) was tested by the twofold serial microdilution method (in 96-well microtiter plates) using Mueller-Hinton Broth medium (Beckton Dickinson, Franklin Lakes, NJ, USA) for bacteria or RPMI-1640 medium for Candida species according to CLSI guidelines [41 ,42 ]. The stock solution of tested compounds was prepared in DMSO and diluted in sterile water. Concentrations of tested agents ranged from 1.56 to 200 μg/mL for bacteria strains and from 1.56 to 100 μg/mL for fungi. The final inoculum of all studied bacteria was 10⁵ CFU/mL (colony forming units per mL) and 0.5–2.5 $\times$ 10⁵ CFU/mL for fungi. Minimal inhibitory concentrations (the lowest concentration of the tested agent that prevents visible growth of a microorganism) were read after 18 h (bacteria) or 24 h (yeasts) of incubation at 35 °C.