Genetic basis of cefiderocol resistance in Acinetobacter baumannii: insights from functional genomics and clinical isolates

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.