Meropenem-Vaborbactam Resistance Selection, Resistance Prevention, and Molecular Mechanisms in Mutants of KPC-Producing Klebsiella pneumoniae

Abstract

Vaborbactam (formerly RPX7009) is a new β-lactamase inhibitor based on a cyclic boronic acid pharmacophore with potent inhibitory activity against Klebsiella pneumoniaecarbapenemases (KPC). It has been developed in combination with meropenem. The objective of these studies was to identify the concentrations of both agents associated with the selection or prevention of single-step mutations leading to reduced sensitivity to the combination and to characterize the selected mutations. Eighteen strains of KPC-producing Klebsiella pneumoniae with various degrees of sensitivity to meropenem (MICs, 8 to 512 μg/ml) and meropenem-vaborbactam (MICs, ≤0.06 to 32 μg/ml) and preexisting resistance mechanisms were selected from a worldwide collection of isolates recovered from surveillance studies, emphasizing strains for which MICs were in the upper range of the meropenem-vaborbactam MIC distribution. Meropenem and vaborbactam at 8 μg/ml each suppressed the drug resistance mutation frequency to <1 × 10^-8 in 77.8% (14/18) of strains, and all strains were inhibited when the meropenem concentration was increased to 16 μg/ml. Mutants selected at lower drug concentrations showed phenotypes associated with previously described carbapenem resistance mechanisms, including ompK36 inactivation in mutants selected from OmpK36-proficient strains and an increased bla_KPC gene copy number in strains with partially functional ompK36 No mutations in the coding region of bla_KPC were identified. These data indicate that the selection of mutants with reduced sensitivity to meropenem-vaborbactam from KPC-producing Klebsiella pneumoniae strains is associated with previously described mechanisms involving porin mutations and the increase in the bla_KPC gene copy number and not changes in the KPC enzyme and can be prevented by the drug concentrations achieved with optimal dosing of the combination.

Keywords: KPC; Klebsiella pneumoniae carbapenemases; resistance development; single-step mutants; vaborbactam.

FIG 1

Rearrangement of the pKpQIL-like plasmid in strains KPM2163 and KPM2164. In KPM2163, an approximately 22-kb region of pKpQIL (nucleotides 7046 to 29059) was duplicated and inserted at nucleotide 2298 and nucleotide 109761. The plasmid had 3 copies of Tn4401. In some cells, the 22-kb copy at nucleotide 2298 and the original 22-kb copy underwent homologous recombination, resulting in the looping out of a 22-kb copy and the 4,747-bp sequence (from nucleotide 2298 to nucleotide 7046) between the 2 copies. The resulting plasmid had 2 copies of Tn4401. In KPM2164, the same 22-kb cassette was duplicated in situ to produce a tandem direct repeat of the 22 kb. The resulting plasmid had 2 copies of Tn4401.