Cefiderocol (CFDC) is a siderophore-conjugated cephalosporin that hijacks bacterial iron uptake pathways to traverse the outer membrane, offering potent activity against carbapenem-resistant Acinetobacter baumannii (CRAB). Although mutations in pirA, a TonB-dependent siderophore receptor, have been linked to CFDC resistance, the broader genetic basis remains poorly defined. Using Himar1 transposon mutagenesis in a CFDC-susceptible sequence type 2 (ST2) CRAB strain, we identified ten genes whose disruption reduced CFDC susceptibility, spanning siderophore-mediated uptake (pirA and puiA), oxidative and redox stress responses (oxyR, nfuA, aarF, cyoA, and bfmRS), and cell envelope morphogenesis (mreB). Most mutants retained wild-type susceptibility to other β-lactams, indicating that reduced CFDC susceptibility can arise independently of target modification. Quantification of cellular iron revealed modest reductions in several mutants, with the largest decreases observed in strains with disruptions in TonB-dependent receptors. Inactivation of pirA or puiA altered the expression of several alternative TonB-dependent siderophore receptors. Whole-genome sequencing of ST2 clinical isolates with reduced CFDC susceptibility uncovered mutations in TonB-dependent receptors, porins, and PBP3, along with increased β-lactamase expression. Importantly, the β-lactamase inhibitor avibactam restored CFDC susceptibility in isolates with β-lactamase upregulation and intact uptake pathways, whereas strains with concurrent uptake defects remained resistant, underscoring the interplay between permeability and enzymatic drug inactivation. These findings define a multifactorial resistance landscape integrating impaired uptake, redox and envelope stress adaptation, and β-lactamase-mediated drug inactivation.IMPORTANCECefiderocol (CFDC) is one of the few remaining antibiotics with activity against carbapenem-resistant Acinetobacter baumannii (CRAB), an urgent global health threat. Yet, resistance to CFDC is increasingly reported, and the underlying mechanisms remain incompletely defined. Most prior studies have examined single pathways, such as loss of TonB-dependent receptors. Here, we used genome-wide transposon mutagenesis together with genomic and phenotypic analysis of CFDC-resistant clinical isolates to generate a more comprehensive view of how resistance emerges. Our findings show that CFDC resistance is multifactorial, involving disrupted siderophore uptake, alterations in oxidative and envelope-stress responses, porin and cell-wall changes, and β-lactamase activity. By defining how these pathways converge, this work provides a broader mechanistic framework for interpreting emerging resistance in clinical settings. These insights underscore the need for integrated surveillance strategies and highlight the biological complexity that must be considered to preserve the effectiveness of this last-line antibiotic.
Keywords: antimicrobial resistance; carbapenem-resistant Acinetobacter baumannii; cefiderocol; comparative genomics; iron metabolism; transposon mutagenesis; whole-genome sequencing.