Avibactam

The pUBYT vector containing bla_KPC-3 under the ISK_pn7 promoter was used as a template for site-directed mutagenesis to create pUBYT containing bla_KPC-2 and bla_KPC-4 with the same promoter (45 (link)). The single point mutation in KPC-2 (Y274H) and double point mutations in KPC-4 (P104R, V240G) were introduced using a QuikChange Lightning site-directed mutagenesis kit (Agilent Genomics) with the primers specified in Table S1 in the supplemental material. Klebsiella pneumoniae Ecl8 was transformed with the resulting pUBYT constructs via electroporation.
S. maltophilia K279a is a well-characterized isolate from Bristol, United Kingdom, and was obtained as previously reported (46 (link)).
MIC values were determined using broth microdilution, in triplicate, in cation-adjusted Mueller-Hinton broth (Sigma) according to the Clinical and Laboratory Standards Institute (CLSI) guidelines (47 ). Experiments were performed in microtiter plates (Corning) containing medium with ceftazidime, imipenem, or aztreonam with inhibitor (4 mg liter⁻¹ avibactam [MedChemExpress] or relebactam [MedChemExpress] dissolved in dimethyl sulfoxide). Plates were incubated overnight at 37°C for 18 to 24 h, and the absorbance at 600 nm was read using a POLARstar Omega (BMG LabTech) plate reader.

Fifteen XDRPA isolates, defined as nonsusceptibility to ≥1 agent in all classes but ≤2 categories including colistin (18 (link)), collected from China-Japan Friendship Hospital from January 2017 to December 2020, were used in this study. The compounds of ceftazidime, avibactam, and imipenem were purchased from MedChemExpress (MCE). These drugs were dissolved in sterile solvents to generate a stock solution, and solutions with various concentrations were further prepared in Mueller-Hinton broth. Others (aztreonam, amikacin, meropenem, piperacillin-tazobactam) were from Wenzhou KONT Biology & Technology Co., Ltd.

β-Lactam antibiotics used were ertapenem (Merck Sharp & Dohme), imipenem (Fresenius Kabi), meropenem (Aurovitas), cefepime (Accord Healthcare), cefoxitin and ceftazidime from Laboratorios Normon (), aztreonam (Bristol-Myers Squibb), as well as doripenem, carbenicillin, piperacillin, ticarcillin, avibactam, relebactam, sulbactam, and tazobactam from MedChem Express. The β-lactam MICs were determined at least in duplicate in strain P. aeruginosa PAO1 following the Clinical and Laboratory Standards Institute (CLSI) broth microdilution method (70 ).

Ceftazidime and avibactam were purchased from MedChemExpress (NJ, USA). Colistin sulfate was purchased from Sigma Aldrich (Saint-Quentin-Fallavier, France). Stock solutions of antibiotics (10,240 mg/L) were prepared in sterile water, stored at −80°C, and diluted in Mueller Hinton Broth II cation adjusted (MHB) on the day of experiment to achieve the desired concentrations.

MICs were determined by the 2-fold serial dilution method in cation-adjusted Mueller-Hinton broth (CAMHB) following the Clinical and Laboratory Standards Institute (CLSI) (59 ). Avibactam (MedChemExpress, China) was used at a fixed concentration of 4 mg/L (60 (link)).