Amikacin

Primary bacterial species identification and antibiotic susceptibility testing were performed with NBPcomb6.23J and Neg MIC6.31J of MicroScan WalkAway Plus (Beckman Coulter, Brea, CA, USA). Strains showing MICs of 4 mg/liter or higher for ceftazidime were judged as potential carbapenemase producers. The MICs were measured using the broth microdilution method in accordance with the CLSI guidelines (M07, 11th edition) (30 ). The following antimicrobial agents were used for antimicrobial susceptibility testing: piperacillin, ceftazidime, and imipenem (Sigma Chemical, St. Louis, MO, USA); ciprofloxacin (LKT Laboratories, St Paul, MN, USA); aztreonam (Tokyo Chemical Industry Co. Ltd., Tokyo, Japan); tazobactam (Toyama Chemical Co., Ltd., Toyama, Japan); meropenem, amikacin, and gentamicin (Wako Pure Chemical Industries, Ltd., Tokyo, Japan); and ertapenem (Merck & Co., Inc., Kenilworth, NJ, USA). The MIC measurement range was 0.125 to 256 mg/liter. E. coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 were used as quality control strains for antibiotic susceptibility testing. The results were interpreted in accordance with the CLSI guidelines (M100, 31st edition) (31 ).

Cefditoren and tebipenem were provided by Meiji Seika Pharma (Tokyo, Japan). Clarithromycin was provided by Taisho Pharmaceutical (Tokyo, Japan). Amoxicillin, amikacin, tobramycin, and gentamicin were purchased from Wako Pure Chemical. Levofloxacin was purchased from LKT Laboratories (St. Paul, MN, USA). A combined effect was defined according to the fractional inhibitory concentration (FIC) index deduced from MIC values, as follows: synergistic effect, FIC ≤ 0.5; additive effect, FIC 0.6–1.0; no effect, FIC 1.1–2; and antagonistic effect, FIC > 2.

MICs of antibiotics and disinfectants were determined by microdilution in LB broth for antibiotics or Mueller-Hinton broth (BD Biosciences) for disinfectants, as described previously (35 (link)). Antibiotic agents, such as colistin, meropenem, amikacin, and ciprofloxacin, and disinfectants, such as ethanol, H₂O₂, SDS, and benzalkonium, were purchased from Fujifilm Wako Pure Chemical Industries. Tigecycline was purchased from the Tokyo Chemical Industry (Tokyo, Japan). The bacterial culture was adjusted to an OD₆₀₀ of 0.001 from the overnight cultures. MICs were monitored using a 2-fold dilution series for each antibiotic or disinfectant.

CST, ampicillin, meropenem, gentamicin, and amikacin were purchased from FUJIFILM Wako Pure Chemical Corp. (Osaka, Japan). Cefotaxime and ciprofloxacin were purchased from Tokyo Chemical Industry (Tokyo, Japan). Minimum inhibitory concentration (MIC)s were determined by the broth microdilution method using cation-adjusted Mueller–Hinton II broth (MHII broth; Becton, Dickinson and Co., Franklin Lakes, NJ) according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI) [20 ]. Isolates with a CST MIC of ≥ 4 mg/L were defined as CST-resistant according to CLSI M100-S32.
Bacterial growth was measured by assessing turbidity (OD₆₀₀) after cultivation for 20 h under the same condition as used for the susceptibility test in the presence of CST (0–128 mg/L). Turbidity was measured using an Infinite M200 PRO instrument (Tecan, Kawasaki, Japan). The skip-well phenomenon was defined as no growth (OD₆₀₀ < 0.1) at a certain CST concentration(s) and growth (OD₆₀₀ > 0.5) at higher CST concentrations. Data are expressed as the means ± standard deviations from independent triplicate experiments.