OBJECTIVE: 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). METHODS: 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. RESULTS: 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. CONCLUSION: 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.
Turowski et al. (Tue,) studied this question.