Genomic and structural insights into the in vivo development of cefiderocol resistance in Klebsiella pneumoniae

Abstract Cefiderocol is a newly developed siderophore-conjugated cephalosporin specifically designed to overcome multidrug-resistant (MDR) Gram-negative bacilli. Nevertheless, reduced susceptibility and resistance to cefiderocol has increasingly been reported, especially derived from alterations in iron transport systems and plasmid-mediated determinants and the expression of structural variants of β-lactamases. Moreover, cefiderocol resistance derived from target alteration remains insufficiently defined. This work aimed to characterize the genomic and structural basis of cefiderocol resistance emerging in vivo during therapy in Klebsiella pneumoniae . Comparative genomics revealed conserved plasmid architecture and no acquisition of novel β-lactamases or iron-uptake determinants. Eighty-two chromosomal variants were identified, among which a missense substitution in ftsI (FtsI G306V), located adjacent to the catalytic serine (S307) of PBP3, emerged as a plausible resistance determinant. Structural modelling suggests that the G306V substitution introduces a significant side-chain volumetric increase, resulting in locally increased steric hindrance within the active-site, possibly impairing cefiderocol interaction with its target. The absence of canonical iron-transport alterations further distinguishes this case from the predominant mechanism described in global molecular epidemiology studies. Large-scale genomic screening revealed a very low prevalence of substitutions affecting ftsI, including in resistance isolates, although possible clonal expansion cannot be ruled out. Our findings align with the broader epidemiological landscape in which cefiderocol resistance is heterogeneous and flexible. Therefore, incorporating analysis of ftsI variants within or proximal to the active-site into genomic surveillance frameworks could improve resistance detection to this last-resort antibiotic. Key points • Resistance occurred without alterations in iron-uptake systems or siderophore pathways. • Mutations in FtsI (G306V) and AcrR (E91K) were identified as key candidates. • FtsI G306V is rare across cefiderocol resistant and global K. pneumoniae genomes.