PROJECT SUMMARY Carbapenem resistant organisms (CRO) have been designated by the CDC as an urgent threat to human health. Specifically, species of Enterobacteriaceae, P. aeruginosa, and A. baumannii that render resistance to carbapenems through the production of degradative β-lactamase enzymes, known as carbapenemases, are associated with high mortality rates and require stringent infection control measures. Unlike non-carbapenemase-producing CROs, CROs that mediate carbapenem resistance through the production of carbapenemases (CPOs) contain plasmids encoding for these degradative enzymes that can be transmitted among pathogenic bacteria, making containment of these organisms of high priority. Further, CPOs are commonly multidrug resistant, increasing the probability of treatment failure. Unfortunately, commercially tests available to detect and characterize CPOs are standalone tests that are not performed until after standard AST results are available. This segmented diagnostic workflow can extend the time to CPO detection to 2-7 days. As a results, CPO-related infections can cost healthcare centers $22,484 to $66,031 per patient. Therefore, a diagnostic test that could be integrated into standard AST panel/systems to offer susceptibility results and CPO confirmation and classification information simultaneously would inform treatment selection and infection control measures days sooner. Herein, we propose the development of a biochemical assay powered by DETECT (Dual- Enzyme Trigger-Enabled Cascade Technology) to offer the first CPO identification and classification test compatible with automated AST systems. DETECT is comprised of targeting and signaling tiers which can be individually modified. The targeting tier features a small molecule probe that mimics the structure of β-lactam antibiotics, with each unique probe displaying distinct recognition for variants and/or classes of these degradative enzymes. We have demonstrated our capacity to synthetically modify the small molecule probe to capture variants of extended spectrum-β-lactamases, and the objectives described herein represent two independent methods of tuning DETECT to capture carbapenemases-producing organisms. If successful, the test formats produced in Aims 1 and 2 would represent unique and/or combination products, which would be optimized for use with a specific AST system in a Phase II project. The final desired test - an AST panel/plate with carbapenemase-targeting DETECT components integrated into the wells, would streamline existing clinical workflows, expedite care delivery to patients, and accelerate infection control de-escalation measures.