Evolved cefiderocol resistance and cross protection - PROJECT SUMMARY/ABSTRACT Pseudomonas aeruginosa (PA) causes lung infections in 44% of the ~31,000 people in the USA with cystic fibrosis (CF). PA CF lung infections can last decades because PA mutates and becomes antibiotic resistant. Antibiotic resistant PA lead to longer hospital stays, higher healthcare costs, and higher mortality rates. Cefiderocol is a new antibiotic approved for the treatment of multidrug resistant infections. Cefiderocol is highly potent because it acts as a trojan-horse: entering bacterial cells through iron-binding siderophore activity and then killing bacteria through inhibition of cell wall synthesis via its cephalosporin antibiotic activity. While highly effective, in some cases cefiderocol has failed during compassionate use treatment of people with CF. Recently we used laboratory experimental evolution to discover 20 different gene mutations in PA that conferred cefiderocol resistance. Among these, cpxS mutations found in >70% of cefiderocol resistant evolved PA also enabled resistant PA to cross-protect susceptible siblings from cefiderocol killing. Notably, cross-protection acted through a novel mechanism that did not involve cefiderocol degradation, but instead was mediated by CPX two- component sensor mutations which led to increased pyoverdine siderophore secretion. Further investigation showed that two PA siderophores had opposing effects on cefiderocol susceptibility: pyoverdine prevented cefiderocol killing, while pyochelin sensitized PA to cefiderocol killing. While the discovery of cross-protective resistant cpxS mutants was troubling, fitness testing showed that cpxS mutants paid significant fitness costs which could lead to the extinction of cpxS strains in drug-free conditions. The central hypothesis of this proposal is that siderophores and fitness costs linked to siderophore production can be coopted to improve cefiderocol potency and limit the development of cefiderocol resistance. The three specific aims of this proposal will investigate 1) CPX-mediated cefiderocol cross-protection mechanisms, 2) Mechanisms driving siderophore-mediated cefiderocol sensitization, and 3) Test cefiderocol cycling to reduce resistance development. In the first aim we will determine how different cpxS variants affect efflux pump expression and siderophore production in PA, and how this in turn affect cross-protection of susceptible wild- type PA by cpxS variants. In the second aim, we will determine how pyochelin sensitizes PA and related Burkholderia species to cefiderocol killing. In the third aim we will use modeling to predict and test how fitness costs paid by cefiderocol resistant strains can be used to cycle cefiderocol treatment and reduce resistance in vitro in a hollow fiber infection model and in vivo in a CF mouse infection model. Altogether these studies will reveal key mechanisms driving cefiderocol cross-protection and inform cefiderocol therapy might be optimized.
