Comprehensive Analysis of a Spontaneous Cefiderocol Resistant Mutant in a <em>Klebsiella pneumoniae </em>KPC Produce

Carbapenem-resistant Klebsiella pneumoniae (CRKP) is an urgent public health threat due to its rapid dissemination and resistance to last-line antibiotics. Cefiderocol (FDC), a novel siderophore cephalosporin, targets resistant Gram-negative pathogens by exploiting bacterial iron uptake mechanisms. However, resistance to FDC is emerging among KPC-producing K. pneumoniae strains. This study characterizes a spontaneous FDC-resistant subpopulation (IHC216) derived from a KPC-producing strain (KPNMA216) using comprehensive genomic, transcriptional, and phenotypic analyses. Given the whole-genome sequencing results, where mutations were identified in genes involved in transcriptional regulation and membrane permeability (ompC) among others, in the present work we further explore their potential implications and conduct a more detailed analysis of the IHC216 genome. qRT-PCR analysis highlighted significant downregulation of iron acquisition systems (fepA, cirA, iroN) and upregulation of alternative iron uptake pathways (iucA, fiU), reflecting a metabolic shift. A notable downregulation of blaKPC-163 correlated with restored susceptibility to carbapenems, indicating collateral susceptibility. Altered expression of pbp2 and pbp3 implicated adaptive changes in cell wall synthesis, potentially affecting FDC resistance mechanisms. Furthermore, enhanced oxidative stress responses via upregulated sodC expression and increased capsule production were observed. These findings underscore the complex interplay of genetic and transcriptional adaptations underlying FDC resistance, highlighting potential therapeutic vulnerabilities.