Project Summary In September 2014, President Barack Obama signed an executive order directing federal resources to improve detection and prevention of antibiotic resistance, with a goal of developing a diagnostic test by 2020 that can distinguish between bacterial and viral infections in at least 20 min. The long-term goal of this research is to provide a novel platform solution for rapid, sensitive and specific detection of pathogenic bacterial infections through development of fluorescent probes specific to individual β-lactamases expressed in the bacteria. β-Lactamases are a class of bacterial hydrolases destroying β-lactam antibiotics and rendering antibiotics resistance in bacterial pathogens. Today many patients with suspected infections are administered antibiotics (most frequently β-lactams) empirically without prior proper identification of the causative agent, resulting in antibiotics overuse and overspread of antimicrobial resistance, with >500,000 deaths in the world annually attributable to these infections. This proposal will focus on two deadly bacteria: Mycobacterium tuberculosis (Mtb) and carbapenem-resistant Enterobacteriaceae (CRE). Tuberculosis represents one of the most dangerous respiratory pathogens in the history of mankind, killing over one million people each year and infecting one third of the world's population. Tubercle bacilli express BlaC, an Ambler class A β-lactamase, so Mtb has the intrinsic resistance to β-lactam antibiotics. Accumulating results including those from us have supported the use of BlaC as the biomarker for Mtb detection. Broad-spectrum carbapenem agents are frequently the last option for effective therapy of infections with antimicrobial resistant organisms, but the emergence of carbapenem-resistant Enterobacteriaceae (CRE) over the past decade has left clinicians with few treatment options. CRE is frequently due to the production of carbapenemase enzymes that efficiently hydrolyze carbapenems and other β-lactam antibiotics. This project will explore novel double-quenching, dual targeting probe design strategy to develop BlaC-specific substrate probes for rapid, highly sensitive Mtb detection (Aim #1) and carbapenemase-specific substrate probes for rapid, highly sensitive CRE detection (Aim #2). These new probes will be evaluated with clinical patient samples. The expected outcome of this research is that a series of novel fluorescent probes will be discovered as a novel assay platform to enable rapid, sensitive and specific detection of Mtb and CRE. We envision that these assays could reduce unnecessary healthcare costs associated with treatment of bacterial infection and prevent further expansion of existing drug-resistant strains.