Influence of a putative TonB-dependent receptor on cefiderocol susceptibility in clinical Enterobacter hormaechei...

To investigate the emergence of resistance to cefiderocol (FDC) in clinical Enterobacter hormaechei isolates and to elucidate the underlying genetic mechanisms, with particular focus on mutations in a putative TonB-dependent receptor (TBDR).

Five non-duplicate clinical FDC-susceptible E. hormaechei isolates were exposed to stepwise increasing concentrations of FDC using disk diffusion and broth microdilution to select for spontaneous FDC-resistant mutants. WGS was performed to identify resistance-associated mutations. Site-directed mutagenesis via CRISPR-Cas9 was employed to confirm causality. Bacterial fitness was evaluated by growth curve experiments under both standard and iron-depleted conditions.

Six spontaneous mutants exhibited increased FDC MICs ranging from 2 to 16 mg/L. All mutants carried distinct mutations in the TBDR gene, including frameshift mutations and premature stop codons. CRISPR-engineered strains harbouring identical mutations displayed the same resistance phenotype, confirming that disruption of TBDR is sufficient to confer increased FDC resistance. Susceptibility to other β-lactams remained unaffected. Growth analyses did not reveal an obvious growth defect in TBDR mutants compared to the parental strain under the tested in vitro conditions, including iron-depleted media.

This study identifies mutations in a putative TBDR as a key mechanism mediating FDC resistance in E. hormaechei. The absence of an obvious growth defect under the tested in vitro conditions suggests that loss of this receptor does not impose a major growth disadvantage. These findings underscore the role of TBDRs in FDC uptake and highlight the potential for resistance development under antibiotic selection pressure through disruption of siderophore-mediated uptake pathways.