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.背景：头孢地尔（Cefiderocol，FDC）是一种铁载体头孢菌素，对碳青霉烯类耐药肺炎克雷伯菌（carbapenem-resistant Klebsiella pneumoniae，CRKP）具有抗菌活性。然而其耐药性已逐渐出现，耐药机制常涉及铁摄取通路与β-内酰胺酶。本研究旨在探讨产NDM的临床分离株在逐步选择性压力下对头孢地尔的适应性机制。方法：一株携带blaNDM-5与blaOXA-181的临床肺炎克雷伯菌分离株（Kp-1），对FDC敏感但对其他受试药物耐药，于37℃、振荡培养条件下在缺铁阳离子调整穆勒-辛顿肉汤（iron-depleted cation-adjusted Mueller–Hinton broth，ID-CAMHB）中传代培养。通过逐步暴露于FDC筛选获得FDC非敏感菌株。对不同代次的菌株进行全基因组测序（whole-genome sequencing，WGS），以追踪变异的出现及频率。为探究早期调控应答，采用实时定量PCR（RT-qPCR）分析17个与头孢地尔耐药、铁摄取及毒力相关的靶基因，比较亲本菌株群（G0）与首次暴露于FDC的代次菌株（G1 + FDC）的基因表达差异。结果：仅暴露于FDC的菌株群积累了突变，突变数量从G1代升至G2代（每株复制品平均为24.3±12.2升至31.7±6.4），非同义突变数量也出现小幅上升（1.7±0.6升至4.7±3.8）。亲本复制品菌株聚类紧密，支持在FDC压力下的从头选择。反复筛选获得baeS（BaeSR双组分系统的感应激酶）的两个非同义替换：V295G与T299P。V295G在各谱系中占比快速升高（例如G01株：G1-FDC代的27.9%升至G2-FDC代的87.9%；G02株：72.5%；G03株：G2-FDC代的74.6%），提示趋同进化。RT-qPCR结果显示，铁摄取/进入通路相关基因（iroN、cirA、fepA、kfu）以及孔蛋白基因ompK35的表达下调，而entB、ompK36、blaNDM-5以及荚膜相关基因（wzm、wbbM）的表达上调；baeS/baeR的转录水平无明显变化。表型实验显示，早期低剂量暴露于头孢地尔并未显著改变荚膜生成量，但生物膜形成能力增强。结论：短期暴露于头孢地尔可重复性地筛选出baeS变异株，并引发转录应答：降低铁载体受体介导的药物摄入途径、重塑外膜，对荚膜影响极小但可增强生物膜生成。BaeS信号通路的非转录调控变化可能改变包膜应激阈值，与受体/孔蛋白平衡改变以及blaNDM-5表达升高协同作用，降低头孢地尔的抗菌活性。上述特征（baeS V295G/T299P、cirA/fepA表达下调）可作为监测标志物，为延缓FDC耐药性的策略提供指导。