OmpK36 GD insertion induces RpoS-dependent blaKPC overexpression in carbapenem-resistant Klebsiella pneumoniae.

Gly115-Asp116 (GD) insertion in the outer membrane porin OmpK36 is prevalent in carbapenem-resistant, KPC-producing Klebsiella pneumoniae, but the molecular mechanisms linking this mutation to enhanced resistance cannot be explained simply by reduced drug permeability. Given that porin mutations compromise bacterial fitness, we investigated the potential role of the stress response regulator RpoS in OmpK36-mediated carbapenemase regulation. Our results demonstrated that among 127 clinical isolates, OmpK36 GD insertion occurred in 50.4% of isolates and was associated with a significantly elevated carbapenem minimum inhibitory concentration and enhanced high-level resistance. In genetically modified strains, the OmpK36 GD insertion impaired bacterial growth and led to a 3.7-fold upregulated rpoS expression and a 2.7-fold increase in blaKPC-2 expression. Bioinformatics analysis identified a putative RpoS binding site in the blaKPC promoter region, which was validated by electrophoretic mobility shift assays. Moreover, RpoS overexpression enhanced blaKPC-2 expression 1.7-fold and increased carbapenem resistance, while disruption of the RpoS binding site reduced blaKPC-2 expression fourfold and decreased resistance levels. Subinhibitory antibiotic treatment upregulated both rpoS and blaKPC-2 expression across multiple strains, further validating a RpoS-mediated regulatory mechanism. These results suggest that OmpK36 GD insertion contributes to carbapenem resistance through RpoS-mediated transcriptional upregulation of blaKPC, revealing a stress-activated regulatory mechanism linking porin structural alterations to modulated carbapenemase expression in K. pneumoniae.