Proteogenomic analysis demonstrates increased <i>bla</i> _OXA-48 copy numbers and OmpK36 loss as contributors to carbapenem resistance in <i>Klebsiella pneumoniae</i>

Antimicrobial resistance arises from complex genetic and regulatory changes, including single mutations, gene acquisitions, or cumulative effects. Advancements in genomics and proteomics facilitate a more comprehensive understanding of the mechanisms behind antimicrobial resistance. In this study, 74 clinically obtained <i>Klebsiella pneumoniae</i> isolates with increased meropenem and/or imipenem MICs were characterized by broth microdilution and PCR to check for the presence of carbapenemase genes. Subsequently, a representative subset of 15 isolates was selected for whole-genome sequencing (WGS) by Illumina and Nanopore sequencing, and proteomic analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) to investigate the mechanisms underlying the differences in carbapenem susceptibility of <i>Klebsiella pneumoniae</i> isolates. Identical techniques were applied to characterize four mutants obtained after sequential meropenem exposure. We demonstrated that in clinically obtained isolates, increased copy numbers of <i>bla</i>_OXA-48-containing plasmids, combined with OmpK36 loss, contributed to high carbapenem MICs without the involvement of OmpK35 or other porins or efflux systems. In the meropenem-exposed mutants, increased copy numbers of <i>bla</i>_CTX-M-15 or <i>bla</i>_OXA-48-containing plasmids, combined with OmpK36 loss, were demonstrated. The OmpK36 loss resulted from the insertion of IS1 transposable elements or partial deletion of the <i>ompK36</i> gene. Additionally, we identified two mutations, C59A and C58A, in the DNA coding the copA antisense RNA of IncFII plasmids and multiple mutations of an IncR plasmid, associated with increased plasmid copy numbers. This study demonstrates that by combining WGS and LC-MS/MS, the effect of genomic changes on protein expression related to antibiotic resistance and the mechanisms behind antibiotic resistance can be elucidated.