Antimicrobial susceptibility test

Antibiotic susceptibilities, including imipenem, meropenem, amikacin, levofloxacin, cefotaxime, aztreonam, amoxicillin– clavulanic acid, and fosfomycin, were determined by the broth microdilution method according to the guidelines provided by the Clinical and Laboratory Standards Institute.12 Minimum inhibitory concentrations (MICs) of tigecycline and colistin were interpreted by EUCAST guidelines (http://www.eucast.org/). MIC by Etest followed the manufacturer’s instructions for some drugs, including cefoperazone/sulbactam, cipro-floxacin, aztreonam, and trimethoprim–sulfamethoxazole. E. coli ATCC 25922 was used as a quality control strain for the antibiotic susceptibility test. The interactions of amoxicillin–clavulanic acid and aztreonam were assessed using the checkerboard method13 with slight modifications. The combinations in the 96-well plates were performed as follows: aztreonam was diluted by twofold dilutions along the x-axis of the plates (from 1/64 MIC to 2 MIC), while amoxicillin–clavulanic acid was diluted by twofold dilutions along the y-axis (from 1/32 MIC to 2 MIC). Subsequently, each well was inoculated with the tested bacterial suspension (5×10⁵ CFU/mL). The plates were then incubated at 37°C overnight. The interaction between aztreonam and amoxicillin–clavulanic acid was determined by quantifying the fractional inhibitory concentration index (FICI) using the following formula: FIC of aztreonam = MIC of aztreonam in combination/MIC of aztreonam alone; FIC of amoxicillin–clavulanic acid = MIC of amoxicillin–clavulanic acid in combination/MIC of amoxicillin–clavulanic acid alone. FICI = FIC_A + FI_CB. “Synergy” was defined when FICI ≤0.5; 0.5 < FICI ≤0.75 means “partial synergy”; 0.76 < FICI ≤1 denotes “additive”; 1 < FICI ≤4 denotes “indifferent”; while “antagonistic” in cases in which the FICI >4.