MIC determination.

MICs were determined by a broth microdilution method according to Clinical and Laboratory Standards Institute (CLSI) recommendations; however, to evaluate the antimicrobial activity of S-649266, the cation-adjusted Mueller-Hinton broth (CAMHB) was supplemented with 20 μM human apo-transferrin (Sigma-Aldrich, St. Louis, MO) (20). The apo-transferrin was added to mimic the ferric-iron-limiting condition in human biological fluids in which free iron is tightly bound to proteins such as transferrin (21). Determination of MIC values in iron-deficient conditions has been used to evaluate the activities of other siderophore antibiotics (14, 16, 17, 22). S-649266 was synthesized at Shionogi & Co., Ltd. (Osaka, Japan). Other antimicrobial agents used in this study were obtained from U.S. Pharmacopeia (Rockville, MD) and include FEP, ceftazidime (CAZ), and MEM and from Sigma-Aldrich (St. Louis, MO), including levofloxacin (LVX). FEP, CAZ, MEM, and LVX were used as reference compounds for MIC determinations against the first set of isolates, while FEP and MEM were used for MIC comparison against the second set of strains. All of the MIC values of the compounds except for S-649266 were measured by a broth microdilution method using CAMHB. The resistance breakpoints of these compounds were defined as recommended by CLSI—16 μg/ml for FEP and CAZ, 4 μg/ml for MEM, and 8 μg/ml for LVX (23).