Occurrence of KPC-71 or the presence of NDM combined with a high expression of

Low-level cefiderocol (CFDC) resistance in carbapenem-resistant Klebsiella pneumoniae (CRKP) represents a growing clinical concern due to its potential for rapid resistance escalation and silent dissemination under antibiotic pressure. This study investigated the molecular epidemiology and resistance mechanisms of low-level CFDC-resistant CRKP in eastern China. Among 382 CRKP clinical isolates, 29 (7.6%) exhibited low-level CFDC resistance. Phylogenetic analysis categorized these isolates into three clusters: Clusters A and B comprised ST11-KL64 strains harboring blaKPC and virulence plasmids, whereas Cluster C primarily consisted of NDM-producing strains from diverse sequence types. Enzyme inhibition assays demonstrated that avibactam significantly reduced CFDC MICs (8-fold to 64-fold) in KPC-producing strains but had a limited impact (2-fold to 4-fold) in strains co-producing KPC and NDM or harboring the KPC-2 variant KPC-71. RT-qPCR showed that blaSHV was the only β-lactamase gene with significantly increased expression. Cloning experiments confirmed that NDM and SHV-12 contributed substantially to CFDC resistance, elevating MICs by 16-fold and 8-fold, respectively. Enzyme kinetics and molecular modeling indicated that KPC-71 enhances CFDC binding via a reduced Ki and a potentially enlarged active-site pocket. These findings suggest that low-level CFDC resistance in CRKP is mainly driven by the presence of KPC-71 or NDM in combination with high blaSHV expression. In particular, a detailed analysis of KPC-71 revealed its critical role in diminishing CFDC susceptibility, offering important insights into the resistance mechanisms of emerging KPC variants.IMPORTANCECefiderocol (CFDC) is a novel siderophore cephalosporin with potent activity against multidrug-resistant gram-negative bacteria, but it has not yet been approved for clinical use in China. The resistance mechanisms of CFDC among clinical isolates in China remain poorly understood, particularly in low-level resistant strains, which may pose a significant threat due to their potential for treatment failure and silent dissemination. In this study, we focused on the resistance mechanisms of low-level CFDC-resistant CRKP strains and revealed distinct resistance patterns that may require tailored treatment strategies. Notably, CFDC-avibactam combinations showed poor efficacy against strains producing NDM or novel KPC variants, whereas they may remain effective against strains producing KPC-2 with high blaSHV expression alone, resulting in ≥8-fold MIC reductions. Moreover, a detailed analysis of KPC-71 uncovered its critical role in reducing CFDC susceptibility, providing important insights into the resistance mechanisms of emerging KPC variants.