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 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 ΔtonB 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 ΔtonB 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.