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

Abstract

Background: Cefiderocol (FDC) is a siderophore cephalosporin active against carbapenem-resistant Klebsiella pneumoniae (CRKP), but resistance has emerged, often involving iron-uptake pathways and β-lactamases. We investigated how an NDM-producing clinical isolate adapts to FDC under stepwise selective pressure.

Methods: A ST147 K. pneumoniae isolate (Kp-1) carrying blaNDM-5 and blaOXA-181, susceptible to FDC but resistant to other agents, was propagated in iron-depleted Mueller-Hinton broth with progressive FDC exposure. Population-based whole-genome sequencing (WGS) tracked mutation dynamics, and RT-qPCR profiled 17 genes related to FDC resistance, iron acquisition, and virulence, comparing the parental population (G0) to the first exposed generation (G1 + FDC).

Results: The mean number of mutations detected in generation G1 under FDC exposure was 24.3 ± 12.2, and in generation G2, it increased to 31.7 ± 6.4. Two nonsynonymous substitutions in baeS (V295G and T299P) were recurrently selected, with V295G reaching ≥ 70% frequency in independent lineages, indicating convergent evolution. RT-qPCR revealed downregulation of iron-uptake and porin genes (iroN, cirA, fepA, kfu, ompK35), and upregulation of entB, ompK36, blaNDM-5, and capsule-related genes (wzm, wbbM); baeS/baeR transcript levels were unchanged. Early FDC exposure enhanced biofilm formation without significantly affecting capsule production.

Conclusions: Short-term FDC exposure reproducibly selects baeS variants and triggers a transcriptional program reducing siderophore-receptor entry and remodeling the outer membrane. These adaptations-together with increased blaNDM-5 expression and biofilm formation-contribute to reduced FDC susceptibility. The recurrent baeS mutations (V295G/T299P) and decreased cirA/fepA expression may serve as early surveillance markers of FDC adaptation in K. pneumoniae.