Intrinsic resistance networks shape cefiderocol susceptibility in ST258 <i>Klebsiella pneumoniae</i>

ABSTRACT 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 β-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 and ldcA ), synthesis ( mrcB and lpoB ), and enterobacterial common antigen biosynthesis ( wzxE and wzyE ), as well as deletion of plasmid-encoded bla KPC-3 , increased CFDC susceptibility. In a CFDC-resistant Δ tonB strain, targeting these envelope homeostasis pathways yielded only limited resensitization relative to the siderophore-competent parental strain. Deletion of bla KPC-3 produced the greatest increase in susceptibility, reducing the CFDC MIC fourfold. This pattern is consistent with a model in which reduced CFDC influx in the Δ tonB background lowers intracellular drug exposure to levels at which the otherwise limited anti-CFDC activity of KPC β-lactamase becomes sufficient to drive resistance. Together, these data support a multi-layered functional network governing cefiderocol susceptibility in ST258 K. pneumoniae , in which iron-dependent drug uptake constrains intracellular exposure, while β-lactamase activity and intrinsic envelope homeostasis pathways further shape bacterial tolerance once cefiderocol enters the cell.