ENAGenomicsChinese Academy of Scienceshttps://www.ebi.ac.uk/ena/browser/view/PRJNA1321917Klebsiella pneumoniaeCarbapenem-resistant Klebsiella pneumoniae (CRKP) poses a major global health threat, particularly in healthcare-associated infections. While carbapenemase- and porin-centered mechanisms are well characterized, how subinhibitory carbapenem exposure selects noncanonical adaptive routes remains unclear. Here, we show that subinhibitory meropenem promotes O_x001E_antigen loss in K. pneumoniae, predominantly mediated by insertion sequences (IS), thereby enhancing carbapenem resistance. O_x001E_antigen deficiency rewires metabolism under meropenem pressure, especially glycine, serine, and threonine pathways, dampening reactive oxygen species (ROS) accumulation and limiting oxidative killing exogenous glycine restores ROS production and meropenem susceptibility. Genomic surveys reveal widespread O_x001E_antigen loss in K. pneumoniae, largely driven by IS, and also in Escherichia coli, and O_x001E_antigen–deficient mutants confirm its role in promoting carbapenem resistance. Importantly, this adaptation entails a trade-off: it improves survival under carbapenem pressure but increases serum susceptibility, destabilizes the capsule, attenuates virulence in murine infection models, and confers collateral sensitivity to aminoglycosides. These findings uncover a previously unrecognized route to carbapenem resistance that links O_x001E_antigen remodeling to metabolic rewiring, offering conceptual and therapeutic leverage points. Overall design: RNA-seq of Klebsiella NK01067 and its ΔwbbM mutant with and without 8 μg/L meropenem.pathogen:prioritypathogen:bacteriumpathogenbacteriaENAfalseO-antigen Deficiency Sparks Metabolic Shifts That Blunt ROS-Mediated Killing by MeropenemO-antigen Deficiency Sparks Metabolic Shifts That Blunt ROS-Mediated Killing by Meropenem2025-09-242025-09-17PRJNA1321917GSE307523573