PROJECT SUMMARY/ABSTRACT: Carbapenem-resistant Klebsiella pneumoniae (CR-Kp) cause life- threatening infections that are associated with unacceptably high mortality rates. CR-Kp is particularly challenging to treat since it often possesses a myriad of molecular resistance determinants that enable it to grow in the presence of most antibiotics. New β-lactam/β-lactamase inhibitors (BL/BLI), such as ceftazidime- avibactam, are not a sustainable therapeutic solution for CR-Kp as monotherapy since there is potential for development of resistance and clinical failure rates remain remarkably high when used alone. Up to 95% of CR- Kp still remain susceptible to at least one of the aminoglycosides (AMG). However, nearly all CR-Kp isolates harbor at least one of the aminoglycoside-modifying enzymes (AME), which inactivate a subset of the AMGs. Thus, selecting an AMG that is tailored to the AMEs and other AMG-resistance determinants (strain-specific AMG) is innovative and provides the foundation for this application, which is focused on development of molecularly precise AMG-based combinations for CR-Kp. Our central hypothesis is that novel combination regimens including short-courses of an optimally dosed, strain-specific AMG and a BL/BLI can maximize killing and resistance suppression of CR-Kp. Our promising preliminary data are highly supportive of our innovative and mechanistic approach. We developed a preliminary model to predict the strain-specific AMG based on an isolate’s AMG-resistance genes. We generated the first data for the combination of the strain-specific AMG with a BL/BLI (ceftazidime-avibactam, meropenem-vaborbactam, and imipenem-relebactam) against CR-Kp in both the hollow fiber infection model (HFIM) and the mouse pneumonia model, where the combination was highly synergistic. We have also developed a novel assay to quantify intracellular AMG concentrations. Leveraging our latest assays, we will elucidate the mechanisms responsible for synergy of the combination between a strain- specific AMG and BL/BLI in CR-Kp to rationally optimize them for future clinical trials. In Aim 1, the precise influence of each AMG-resistance determinant on AMG activity will be defined and novel predictors of AMG- resistance will be elucidated. In Aim 2, we will design novel short-course AMG treatment regimens that are efficacious. We will define the time-course of AMG-tolerance and resistance emergence using systems pharmacology and protein synthesis assays. In Aim 3, we will rationally optimize AMG and BL/BLI combinations by identifying mechanisms of synergy and assessing antibiotic target site concentrations. The HFIM will be used to define the pharmacodynamics of combinations and determine optimal timing of antibiotic administration. In Aim 4, we will develop quantitative and systems pharmacology (QSP) models that integrate our mechanistic data and rationally optimize AMG-based combination dosing strategies based on AMG-resistance determinants in CR-Kp. QS...