Impact of Cefiderocol on Klebsiella pneumoniae : A Population-based Longitudinal Analysis of Phenotypic and Genotypic Responses at Subinhibitory and Inhibitory Concentrations

Background: Cefiderocol (FDC) is a siderophore cephalosporin active against carbapenem-resistant Klebsiella pneumoniae (CRKP). However, resistance has emerged, often implicating iron-uptake pathways and β-lactamases. We investigated how an NDM-producing clinical isolate adapts to cefiderocol under stepwise selective pressure.Methods: A clinical K. pneumoniae isolate (Kp-1) carrying blaNDM-5 and blaOXA-181, susceptible to FDC but resistant to other tested agents, was propagated in iron-depleted cation-adjusted Mueller–Hinton broth (ID-CAMHB) at 37 °C with agitation. FDC non-susceptibility was selected by progressive exposure. Population whole-genome sequencing (WGS) was performed longitudinally to track variant emergence and frequency. To probe early regulatory responses, RT-qPCR profiled 17 target genes linked to cefiderocol resistance, iron acquisition, and virulence, comparing the parental population (G0) to the first FDC-exposed generation (G1 + FDC).Results: Only FDC-exposed populations accumulated mutations, which increased from G1 to G2 (mean 24.3±12.2 to 31.7±6.4 per replicate), with a modest rise in nonsynonymous changes (1.7±0.6 to 4.7±3.8). Ancestral replicates clustered tightly, supporting de novo selection under FDC. Recurrently, two nonsynonymous substitutions in baeS (sensor kinase of BaeSR) were selected: V295G and T299P. V295G rose sharply across lineages (e.g., G01: 27.9% in G1-FDC to 87.9% in G2-FDC; G02: 72.5%; G03: 74.6% in G2-FDC), indicating convergent evolution. RT-qPCR revealed downregulation of iron-uptake/entry pathways (iroN, cirA, fepA, kfu) and the porin ompK35, with upregulation of entB, ompK36, blaNDM-5, and capsule genes (wzm, wbbM); baeS/baeR transcript levels were unchanged. Phenotypically, early and low exposure to cefiderocol did not significantly change capsule production, whereas biofilm formation increased.Conclusions: Short-term cefiderocol exposure reproducibly selects baeS variants and a transcriptional response that reduces siderophore-receptor entry and remodels the outer membrane, with minimal impact on capsule but increased biofilm production. Non-transcriptional changes of BaeS signaling likely shifts the envelope-stress set-point, cooperating with altered receptor/porin balance and elevated blaNDM-5 to reduce cefiderocol activity. These signatures (baeS V295G/T299P, reduced cirA/fepA expression) may serve as surveillance markers and guide strategies to delay FDC resistance.