I am an Assistant Professor of Pediatrics at The Johns Hopkins University School of Medicine and am very committed to improving my understanding of the mechanisms of Gram-negative resistance so I can ultimately become a future leader in developing, validating, and implementing rapid diagnostics for the detection of multidrug-resistant Gram-negative organisms (MDRGNs). In 2013, the Centers for Disease Control and Prevention assigned the highest threat level to carbapenemase- producing Enterobacteriaceae (CPE), declaring they require urgent public health attention. CPE are endemic in the US mid-Atlantic region, representing 13% of clinical isolates of healthcare-acquired Klebsiella pneumoniae. CPE bacteremia are associated with mortality upwards of 60%. Improving outcomes of patients with CPE bacteremia requires their prompt detection to ensure patients receive optimal antibiotic therapy as early as possible. There is no standardized, easy-to-use, reliable assay for carbapenemase detection. Most diagnostic laboratories do not test for carbapenemase production, potentially leading to poor patient outcomes. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) is a technology used to detect microorganisms that has become increasingly commonplace in diagnostic laboratories. It is rapid, cost-effective, and accurate. Species and genus identification are available in as little as 10 minutes from the time bacterial growth is detected in sterile specimens. MALDI TOF is currently not being used for the identification of antibiotic resistance in clinical practice. We believe we can capitalize on this FDA-approved technology to detect carbapenemases directly from blood culture broths at least 27 hours earlier than existing methods. Although the distribution of carbapenemases endemic to the mid-Atlantic US has not been previously characterized, it is estimated that approximately 50% of carbapenemases are KPC-3 carbapenemases. Our preliminary data indicate that MALDI TOF can be successfully used to identify the presence of KPC-3 carbapenemases. We believe that we can expand upon this and build a comprehensive MALDI TOF library to rapidly detect the wide variety of carbapenemases endemic to the US. The overarching goals of this proposal are to (a) identify the organism and carbapenemase-encoding gene combinations in a highly endemic region for CPE using existing isolates from 8 hospitals; (b) develop a comprehensive MALDI TOF library for the rapid detection of carbapenemases directly from positive bloodstream isolates with the goal that this library can eventually be exported to clinical microbiology laboratories across the country for rapid detection of CPE; and (c) determine the accuracy and potential impact of MALDI TOF on optimizing antibiotic therapy for patients admitted to any of 5 hospitals in the Hopkins Health System with CPE bacteremia. Because the K23 mechanism does not allow for experimental diagnostics to directly impact patient care, the results of the proposed method in Aim 3 will not be used to inform decisions regarding patient care but will lay the groundwork for a future R01 interventional study. For me to be able to continue as a clinician investigator, receiving a Career Development Award is essential. Because of the extremely busy clinical pediatric infectious diseases service at Johns Hopkins Hospital, I currently devote approximately 70% effort to clinical and administrative responsibilities. Without the protected time for additional training to advance my research career afforded by a K award, it would be extremely difficult to conduct effective and meaningful research. Furthermore, I want to change my focus from clinical outcomes studies to understanding how to enhance rapid diagnostic assays to improve the detection of MDRGNs. Since I do not have a basic science background, a Career Development Award would afford me time to improve my understanding of available assays for both phenotypic and genotypic detection of MDRGNs so that I can ultimately become a leader in the field of rapid diagnostics for MDRGNs. Without a clear understanding of the mechanisms of Gram-negative resistance, I believe I will only have a superficial understanding of the Gram- negative crisis. Although I have begun to explore training in molecular diagnostics of Gram-negative bacteria this past year, the laboratory methodology involved in my proposal is labor intensive and entails multiple complex steps. I would benefit from further mentorship to be able to function independently in the laboratory setting. I am very optimistic about my proposed future path. Developing a strong foundation in laboratory methods for identifying Gram-negative resistance is critical for me to become an independent clinician investigator in developing, validating, and implementing rapid diagnostics assays for the detection of MDRGN organisms.