PROJECT SUMMARY / ABSTRACT Diabetic foot ulcers (DFUs) remain one of the most common complications of diabetes mellitus (DM) and are the leading cause of lower extremity amputation with every sixth individual suffering an early demise as a result. Many complicating factors of DM contribute and interrupt normal physiologic healing processes. Five-year mortality after DFU occurrence is 40% and is 10-fold higher than non-diabetic cohorts. Despite major scientific advances in the understanding of wound healing physiology, only one-half of DFUs heal when standard of care is met. This unfortunate truth highlights the need to identify modifiable risk factors for healing that may be the cause of non-healing events in the treatment of DFUs. The DFU microbiome may represent these important modifiable risk factors in obtaining wound healing. The DFU microbiome is comprised of the genetic material of all the microbiota that cohabitate the DFU. Past observational studies have implicated one or more of these microbiotas as contributors to non-healing, but these studies have suffered from significant design limitations and lack consensus assessment. Most studies were only cross-sectional in design and focused primarily on non-infected DFUs. Furthermore, these studies did not collect biospecimens consistently across studies. In this career development, we propose to understand the role of the microbiome in the pathogenesis of DFU and understand the trajectories of the microbiome across DFU wound progression in three aims. Aim 1 will perform a cross-sectional cohort of patients with DFU to compare the microbiome of those with DFI to infection-free DFUs using metagenomics next generation sequencing (mNGS). Aim 2 will leverage this initial cohort to longitudinally determine the temporal relationships and interactions between pathogen presence, wound healing, and development of infection. Aim 3 will assess the clinical feasibility of using mNGS to predict antibiotic therapy against identified pathogens in infected DFU in a pilot clinical trial. The overall goal of this project is to identify modifiable risk factors in the DFU microbiome that will lead to interventional clinical trials to prevent and/or treat DFUs. This proposal is essential to my career development. I will become an independent clinical researcher with expertise in diabetic foot complications. The formal training in clinical trial execution and biostatistics will provide practical experiences and will set the stage for successful completion of not only this project, but also of future investigations. The clinical trial component of my career development will allow me to take the results from this study and seamlessly transition into interventional studies that will lead to new treatments for patients suffering from DFUs. Drs. Rodica Pop-Busui and Keith Kaye are ideally suited as mentors for this project with their complementary expertise in diabetes, neuropathy, infectious disease, and clinical t...