Spontaneous Emergence of Cefiderocol Resistance in Klebsiella pneumoniae KPC-163: Genomic and Transcriptomic Insights

<b>Background/Objectives:</b> Carbapenem-resistant <i>Klebsiella pneumoniae</i> (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 <i>Klebsiella pneumoniae</i> carbapenemase (KPC)-producing <i>K. pneumoniae</i> strains. This study characterizes a spontaneous FDC-resistant subpopulation (IHC216) derived from a KPC-producing strain (KPNMA216) using comprehensive genomic, transcriptional, and phenotypic analyses. <b>Methods</b>: Given the whole-genome sequencing results, where mutations were identified in genes involved in transcriptional regulation and membrane permeability (<i>ompC</i>) among others, in the present work we further explore their potential implications and conduct a more detailed analysis of the IHC216 genome. A qRT-PCR analysis highlighted significant downregulation of classical siderophore-mediated iron acquisition systems (<i>fepA</i>, <i>cirA</i>, <i>iroN</i>) and upregulation of alternative iron uptake pathways (<i>iucA</i>, <i>fiU</i>), reflecting a switch in iron acquisition strategies. <b>Results</b>: A notable downregulation of <i>bla</i>_KPC-163 correlated with restored susceptibility to carbapenems, indicating collateral susceptibility. Altered expressions of <i>pbp2</i> and <i>pbp3</i> implicated adaptive changes in cell wall synthesis, potentially affecting FDC resistance mechanisms. Furthermore, enhanced oxidative stress responses via upregulated <i>sodC</i> expression and increased capsule production were observed. <b>Conclusions</b>: These findings underscore the complex interplay of genetic and transcriptional adaptations underlying FDC resistance, highlighting potential therapeutic vulnerabilities.