In vitro selection of aztreonam/avibactam resistance in dual-carbapenemase-producing Klebsiella pneumoniae

Objectives: To examine the in vitro selection of aztreonam/avibactam resistance among MBL-producing Klebsiella pneumoniae and to understand the mechanism of increased resistance. Methods: The MICs of aztreonam were determined with and without avibactam (4 mg/L) using a broth microdilution method. Single-step and multi-step mutant selection was conducted on five MBL-producing K. pneumoniae strains, including two dual carbapenemase producers. Genomic sequencing and gene cloning were performed to investigate the mechanism of increased resistance. Results: We examined the MICs for 68 MBL-producing K. pneumoniae isolates, including 13 dual carbapenemase producers. Compared with aztreonam alone, the addition of avibactam (4 mg/L) reduced the MICs for all isolates by >128-fold, with MIC₅₀ and MIC₉₀ values of 0.25 and 1 mg/L, respectively. One NDM-1-, OXA-48-, CTX-M-15-and CMY-16-positive ST101 K. pneumoniae strain was selected to be resistant to aztreonam/avibactam, with a >16-fold increase in MIC (>128 mg/L). WGS revealed that the resistant mutants lost the bla_NDM-1 gene, but acquired amino acid substitutions in CMY-16 (Tyr150Ser and Asn346His). Construction of bla_CMY-16 mutants confirmed that the substitutions (Tyr150Ser and Asn346His) were primarily responsible for the decreased susceptibility to aztreonam/avibactam. In addition, transfer of bla_CMY-16 mutant (Tyr150Ser and Asn346His) plasmid constructs into certain clinical carbapenemase-producing isolates demonstrated >64-fold increased MICs of aztreonam/avibactam and aztreonam/avibactam/ceftazidime. Conclusions: Aztreonam in combination with avibactam showed potent in vitro activity against MBL-producing K. pneumoniae. However, our study suggested the likelihood of aztreonam/avibactam resistance among MBL-and AmpC-co-producing strains and clinical practice should beware of the possibility of the emerging resistance.