Colistin

The minimum inhibitory concentrations (MICs) of common antibiotics (National Institutes for Food and Drug Control, Beijing, China) against ESBL-producing E. coli were determined using the broth microdilution method with E. coli ATCC 25922 as the control strain. Interpretation of results followed the criteria of CLSI 201328 . The interpretation breakpoint of cefoperazone/Sulbactam (Pfizer Inc., USA) was extrapolated from that of cefoperazone, and those of tigecycline and colistin (Pfizer Inc.) were based on the criteria of the European Committee on Antimicrobial Susceptibility Testing (EUCAST-2012).

PAβN and CCCP inhibitory tests were used to explore the role of efflux pumps in both the colistin- and tigecycline-resistant strains (Ni et al., 2016 (link)). MIC values for colistin and tigecycline (Pfizer, NY, United States) were determined using the broth microdilution method as recommended by the CLSI guidelines (Clinical and Laboratory Standards Institute [CLSI], 2015 ). Briefly, stock solutions of PAβN (Sigma-Aldrich, Shanghai, China) and CCCP (Sigma-Aldrich, Shanghai, China) were prepared at 10 mg/mL in sterile water and DMSO, respectively. Bacterial growth in Mueller-Hinton broth containing tigecycline or colistin with and without PAβN (25 mg/L) or CCCP (10 mg/L) was then evaluated in parallel. A growth control with 25 mg/L PAβN or 10 mg/L CCCP in Mueller-Hinton broth was added to evaluate the effect of each of the efflux pump inhibitors on their own in each strain.