Project Summary The rise of antibiotic resistance among healthcare associated pathogens has created a public health emergency and is a major challenge to the provision of effective medical care. Multidrug-resistant (MDR) Pseudomonas aeruginosa is labeled as a serious threat by the Centers for Disease Control and Prevention, and disproportionately impacts medically vulnerable patients such as those in the intensive care unit. The emergence of resistance to front-line antibiotics, including carbapenems and recently introduced β-lactam/β- lactamase inhibitor combinations, has led to the use of alternate regimens with decreased efficacy and increased toxicity. Cefiderocol (FDC) is a novel siderophore cephalosporin that retains in vitro activity against MDR-P. aeruginosa. This antibiotic mimics the iron binding molecules P. aeruginosa requires for growth, and is actively transported into the periplasmic space by proteins on the outer membrane of the bacteria called TonB dependent receptors (TBDR). Resistance to FDC has been linked to a loss of expression of these TBDRs in clinical isolates of P. aeruginosa. Further, an analysis of genomes of P. aeruginosa collected before the approval of FDC found that mutations predicted to lead to decreased expression of TBDR genes were present in carbapenem-resistant isolates. Using population analysis profiles (PAP), several strains of P. aeruginosa with TBDR mutations showed heteroresistance to FDC, or growth of a small population of bacteria at antibiotic concentrations above the clinical resistance breakpoint despite testing susceptible on standard susceptibility testing. In two major specific aims, this proposal will investigate the hypothesis that mutations in TBDR genes are present across clinical isolates of P. aeruginosa from diverse geographic areas and lead to the emergence of resistance on exposure to FDC. In the first aim, the prevalence of TBDR gene mutations will be evaluated in a collection of nearly one thousand carbapenem-resistant P. aeruginosa from the Prospective Observational Pseudomonas study (POP). Alterations in this pathway will be linked to FDC susceptibility testing results, an assessment of heteroresistance using PAP, and clinical features of P. aeruginosa colonization and infection. In the second aim, we will investigate strategies to understand and mitigate the emergence of resistance associated with TBDR mutations. First, a disk diffusion-based method to identify heteroresistant isolates will be developed as a screening test for the clinical microbiology laboratory. Second, a hollow fiber infection model simulating human pharmacokinetics of FDC will be used to evaluate the emergence of FDC resistance at clinically relevant drug concentrations. This proposal will provide important information on the distribution and prevalence of mutations associated with resistance to FDC in P. aeruginosa. The development of an assay to identify isolates at risk of resistance, and an understanding of...