Loss of the ferripyochelin receptor FptA drives reduced cefiderocol susceptibility and impairs fitness in <i>Pseudomonas aeruginosa</i> PA14

<i>Pseudomonas aeruginosa</i> is an opportunistic human pathogen and a frequent cause of multidrug-resistant infections. This organism continues to evade antimicrobial therapy despite the clinical introduction of new antipseudomonal antibiotics over the past several years. One of these agents is cefiderocol (FDC), a novel siderophore-cephalosporin conjugate antibiotic that was designed to overcome both intrinsic and acquired β-lactam resistance mechanisms in <i>P. aeruginosa</i>. However, studies have demonstrated that inactivation of energy transducer protein (TonB)-dependent receptors, most notably the catechol siderophore receptor <i>piuA,</i> can substantially curtail the drug's ability to permeate the bacterial outer membrane, leading to rapid development of resistance. In this study, we examined the FDC resistance mechanisms of the laboratory strain PA14. We demonstrated that inactivation of the ferripyochelin receptor FptA was a first-step mutation toward FDC resistance. Through transposon mutagenesis, we identified several resistance pathways following <i>fptA</i> inactivation, such as the loss of an additional FDC receptor and overexpression of the MuxABC-OpmB multidrug efflux system. Introduction of clinically identified mutations analogous to these transposon insertions in the absence of <i>fptA</i> conferred full FDC non-susceptibility while preserving the activity of other antipseudomonal β-lactam antibiotics. We also demonstrated that inactivation of <i>fptA</i> in a pyoverdine biosynthetic mutant disrupted bacterial iron homeostasis and conferred a fitness disadvantage. These FDC resistance mechanisms identified in PA14 highlight the long-term challenges of using FDC treatment for drug-resistant <i>P. aeruginosa</i> infections.