In vitro-obtained meropenem-vaborbactam resistance mechanisms among clinical Klebsiella pneumoniae carbapenemase-producing K. pneumoniae isolates

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A novel ß-lactam-β-lactamase inhibitor (BLBI), meropenem (MEM), combined with the boronate-based inhibitor vaborbactam (VAB), has recently been introduced for the treatment of infections caused by Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacterales. The purpose of this study was to select for MEM-VAB resistance using a collection of eight KPC-producing K. pneumoniae clinical isolates, including three that produce KPC variants conferring ceftazidime-avibactam (CAZ-AVI) resistance, and subsequently decipher the corresponding resistance mechanisms. Mutants were selected in a stepwise process on agar plates containing different MEM-VAB concentrations. Susceptibility testing was performed by broth microdilution, and complementation assays were performed with wildtype ompK36. Whole genome sequencing was performed on mutants, and KPC copy number was assessed by quantitative polymerase chain reaction . Mutants were obtained from 6/8 tested isolates and reduced susceptibility to all tested β-lactams, and BLBIs, including CAZ-AVI, imipenem-relebactam, and aztreonam-AVI, were observed. No mutations were identified in the blaKPC. However, mutations in ompK36 were observed in four mutant lineages, and complementation with a wild-type ompK36 resulted in a reduction of minimal inhibitory concentrations to both MEM-VAB and other ß-lactams/BLBIs. blaKPC gene copy numbers were significantly increased in four mutant lineages. Whole genome sequencing identified genomic rearrangements in two lineages comprising mutations in the plasmid replicon encoding gene and duplication of the Tn4401 transposon bearing the blaKPC gene into a ColE-like, high copy number plasmid. In contrast to what is observed with KPC-producing mutants exhibiting resistance to CAZ-AVI, mainly corresponding to mutated KPC enzymes, here the MEM-VAB-resistant mutants showed permeability defects combined with increased KPC production, resulting from genomic rearrangement.