Avibactam

Antimicrobial susceptibility testing was performed by reference broth microdilution methods conducted according to Clinical and Laboratory Standards Institute (CLSI) procedures [14 ]. Quality control testing was performed daily to ensure proper test conditions. Quality control strains included E coli ATCC 25922 and NCTC 13353, K pneumoniae ATCC 700603, ATCC BAA-1705 and BAA-2814, and Pseudomonas aeruginosa ATCC 27853. CLSI guidelines were used for the interpretation of susceptibility rates, with the exception of tigecycline, for which US Food and Drug Administration (FDA) breakpoints were applied [15 , 16 ]. Avibactam was provided by Allergan. Other agents were acquired from Sigma-Aldrich (St Louis, Missouri), US Pharmacopeia (Rockville, Maryland), Advanced Chemblocks (Hayward, California; relebactam), or MedChemExpress (Monmouth Junction, New Jersey; vaborbactam).

Antimicrobial susceptibility testing (AST) of the clinical isolates PA_HTX1 and PA_HTX2 was performed in the clinical laboratory using a Microscan Walk-Away and E-test (for colistin, C/T and ceftazidime/avibactam [CZA]). Synergy testing was performed by applying an aztreonam (ATM) or meropenem (MEM) E-test strip to Mueller-Hinton agar plates containing either ceftazidime plus avibactam (Allergan), at a final concentration of 2.2 µg/mL of the avibactam component, or vaborbactam alone (The Medicines Co.) at 2 µg/mL. This concentration was selected to mimic the serum nadir of avibactam and vaborbactam from human pharmacokinetic/pharmacodynamic data [13–17 (link)]. The AST for the E coli mutants was performed in triplicate by normalizing strains to an optical density (OD)₆₀₀ of 0.08 in Mueller-Hinton II broth, inducing with 1 mM IPTG for 2 hours and normalizing again to a 0.5 McFarland standard (OD_600nm 0.08–0.1), before plating on Mueller-Hinton agar plates containing 40 µL of 100 mM IPTG. E-test strips (bioMérieux) were applied for all antibiotics tested, and MICs were read after 24 hours of incubation at 37°C.