Intrinsic resistance networks shape cefiderocol susceptibility in ST258 Klebsiella pneumoniae

Cefiderocol (CFDC) is a siderophore-conjugated cephalosporin developed to overcome multidrug resistance in Gram-negative bacteria. Despite its unique iron-dependent entry mechanism, CFDC resistance has emerged in Klebsiella pneumoniae, primarily driven by alterations in siderophore transport and {beta}-lactamase evolution; however, the broader intrinsic resistome that supports CFDC tolerance remains incompletely defined. Here, we performed high-density transposon mutagenesis (Tn-Seq) in the epidemic ST258 K. pneumoniae to map the functional genetic landscape of CFDC susceptibility. Tn-Seq identified siderophore uptake components (tonB and cirA) as the dominant determinants of CFDC resistance. In contrast, disruption of genes involved in peptidoglycan recycling (ampG, ldcA), synthesis (mrcB, lpoB) and enterobacterial common antigen (ECA) biosynthesis (wzxE, wzyE), as well as deletion of plasmid-encoded blaKPC-3, increased CFDC susceptibility. In a CFDC-resistant {Delta}tonB strain, targeting these envelope homeostasis pathways yielded only limited resensitization relative to the siderophore-competent parental strain. Deletion of blaKPC-3 produced the greatest increase in susceptibility, reducing the CFDC MIC four-fold. This pattern is consistent with a model in which reduced CFDC influx in the {Delta}tonB background lowers intracellular drug exposure to levels at which the otherwise limited anti-CFDC activity of KPC {beta}-lactamase becomes sufficient to drive resistance. Together, these data define a hierarchical genetic architecture for CFDC resistance in ST258 K. pneumoniae, in which iron-dependent drug uptake is primary, {beta}-lactamase activity is secondary, and intrinsic envelope stress buffering shapes bacterial fitness once CFDC enters the cell.