Mechanisms of Cefiderocol Resistance in Carbapenemase-Producing <em>Enterobacteriaceae</em>: Insights from Comparative Genomics

Background/Objectives: Cefiderocol is a novel siderophore cephalosporin with potent in vitro activity against a broad spectrum of Gram-negative bacteria, including carbapenemase-producing Enterobacteriaceae CPE). However, the recent emergence of re-sistance in clinical settings raises important concerns regarding its long-term effective-ness. This study aims to investigate the genomic determinants associated with cefiderocol resistance in CPE isolates of human origin. Methods: Comparative genomic analyses were conducted between cefiderocol-susceptible and -resistant CPE isolates recovered from human clinical and epidemiological samples at a tertiary care hospital. Whole-genome sequencing, variant annotation, structural modeling, and pangenome analysis were performed to characterize resistance mechanisms. Results: A total of 59 isolates (29 resistant and 30 susceptible) were analyzed, predominantly comprising Klebsiella pneumoniae, Escherichia coli, and Enterobacter cloacae. The most frequent car-bapenemase among resistant isolates was blaNDM, which was also present in a subset of susceptible strains. Resistant isolates exhibited a significantly higher burden of non-synonymous mutations in siderophore receptor genes, notably within fecR, fecA, fiu, and cirA. Structural modeling predicted deleterious effects for mutations such as fecR:G104S and fecA:A190T. Additionally, porin loss and loop 3 insertions (e.g., GD/TD) in OmpK36, as well as OmpK35 truncations, were more frequent in resistant isolates, partic-ularly in high-risk clones such as ST395 and ST512. Genes associated with toxin&ndash;antitoxin systems (chpB2, pemI) and a hypothetical metalloprotease (group_2577) were uniquely found in the resistant group. Conclusions: Cefiderocol resistance in CPE appears to be multifactorial. NDM-type metallo-&beta;-lactamases and mutations in siderophore up-take systems&mdash;especially in the fec, fhu, and cir operons&mdash;play a central role. These may be further potentiated by alterations in membrane permeability, such as porin disruption and efflux deregulation. The integration of genomic and structural approaches provides valuable insight into emerging resistance mechanisms and may support the development of diagnostic tools and therapeutic strategies.