The minimum inhibitory concentrations (MICs) were determined after 18 h of incubation at 37°C by dilution on Sensitivity Disc Agar-N (Nissui Pharmaceuticals, Tokyo, Japan). The following antimicrobial agents were tested: piperacillin, piperacillin/tazobactam (Toyama Chemicals, Tokyo, Japan), imipenem, amikacin (Banyu Pharmaceuticals, Tokyo, Japan), meropenem (Dainippon-Sumitomo Pharmaceuticals, Tokyo, Japan), aztreonam (Eisai Co., Tokyo, Japan), ceftazidime (Glaxo Japan Co., Tokyo, Japan), tobramycin (Shionogi Pharmaceuticals, Tokyo, Japan), arbekacin (Meiji Seika Co., Tokyo, Japan), ciprofloxacin (Bayer Japan Co., Tokyo, Japan) and colistin (Wako Junyaku Co., Osaka, Japan). These antibiotics except for ciprofloxacin were provided in the form of a freeze-dried amorphous powder. The inocula (10
4 colony-forming units [cfu]/spot) were plated using a multipoint inoculator (Sakuma Co., Tokyo, Japan). The MIC was defined as the lowest drug concentration that inhibited visible growth.
P. aeruginosa IFO 3919 was used as the reference strain. The drug concentrations (breakpoints) were set as follows: piperacillin, 64 mg/l; imipenem and meropenem, 8 mg/l; aztreonam and ceftazidime, 16 mg/l; amikacin, tobramycin and arbekacin, 4 mg/l; ciprofloxacin and colistin, 2 mg/l. Breakpoints used for all agents (except for amikacin, tobramycin, arbekacin and colistin) were according to the National Committee for Clinical Laboratory Standards (NCCLS) criteria [
14]. The concentration of amikacin and tobramycin was 4 mg/l, which is lower than the criteria of the NCCLS. This was because in Japan, the routine dose of these agents is lower (ex. in the case of amikacin, 200–400 mg/day in one to two divided doses) than that in Western countries. The concentration of colistin used was according to a report by Soussy
et al [
15].