Pentamidine

Clinical isolates were obtained from a tertiary teaching hospital (Wenzhou, China). Reference strains (E. coli ATCC25922, K. pneumoniae ATCC700603, and A. baumannii ATCC19606) were used in all mechanism assays. Antibiotics including rifampicin, linezolid, erythromycin, tetracycline, vancomycin, and polymyxin B (PMB) (Kangtai, Zhejiang, China) were used in this study. Pentamidine, carbonyl cyanide m-chlorophenylhydrazone (CCCP), and their solvent dimethyl sulfoxide (DMSO) were purchased from MedChemExpress (Junction, NJ, USA) and Sigma-Aldrich, USA (St. Louis, MO, USA). The broth microdilution (BMD) method was used to determine the MICs of all agents against all bacteria (Table S2). Sub-MICs of PMB (0.0625 mg/L) or SDS (0.01% v/v) (Solarbio, Beijing, China) were used as membrane permeators and CCCP (5 mg/L) as an efflux inhibitor [21 (link)]. Sub-MICs of CCCP (5 mg/L) and PMB (0.0625 mg/L) were added to serially two-fold-increased concentrations of linezolid, rifampicin, or vancomycin to determine their effects on the MICs of these antibiotics for multiple GNBs.

Pentamidine (MedChemExpress Co., Ltd., USA) was initially dissolved in dimethyl sulfoxide (DMSO) (Sigma-Aldrich, USA) (final concentration of DMSO ≤2.5% [vol/vol]) (25 (link)). Linezolid (Kangtai, Wenzhou, China) was initially dissolved in sterile distilled water and was subsequently dissolved in CAMHB (Sigma-Aldrich, USA) to obtain the desired drug concentrations.