Mechanisms of Cefiderocol Resistance in Carbapenemase-Producing Enterobacterales: Insights from Comparative Genomics

<b>Background/Objectives</b>: Cefiderocol is a novel siderophore cephalosporin with potent in vitro activity against a broad spectrum of Gram-negative bacteria, including carbapenemase-producing Enterobacterales (CPE). However, the recent emergence of resistance in clinical settings raises important concerns regarding its long-term effectiveness. This study aims to investigate the genomic determinants associated with cefiderocol resistance in CPE isolates of human origin. <b>Methods</b>: 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 modelling, and pangenome analysis were performed to characterize resistance mechanisms. <b>Results</b>: A total of 59 isolates (29 resistant and 30 susceptible) were analyzed, predominantly comprising <i>Klebsiella pneumoniae</i>, <i>Escherichia coli</i>, and <i>Enterobacter cloacae</i>. The most frequent carbapenemase gene among the resistant isolates was <i>bla</i>_NDM, which was also present in a subset of susceptible strains. The resistant isolates exhibited a significantly higher burden of non-synonymous mutations in their siderophore receptor genes, notably within <i>fecR</i>, <i>fecA</i>, <i>fiu</i>, and <i>cirA</i>. Structural modelling predicted deleterious effects for mutations such as <i>fecR:</i>G104S and <i>fecA:</i>A190T. Additionally, porin loss and loop 3 insertions (e.g., GD/TD) in OmpK36, as well as OmpK35 truncations, were more frequent in the resistant isolates, particularly in high-risk clones such as ST395 and ST512. Genes associated with toxin-antitoxin systems (<i>chpB2</i>, <i>pemI</i>) and a hypothetical metalloprotease (group_2577) were uniquely found in the resistant group. <b>Conclusions</b>: Cefiderocol resistance in CPE appears to be multifactorial. NDM-type metallo-β-lactamases and missense mutations in siderophore uptake systems-especially in those encoded by <i>fec</i>, <i>fhu</i>, and <i>cir</i> operons-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 insights into emerging resistance mechanisms and may support the development of diagnostic tools and therapeutic strategies.