Chronic, non-healing wounds are common and costly complications of diabetes. Microbial colonization and biofilm formation are hypothesized to impair wound healing and contribute to severe complications such as osteomyelitis and amputation. Although all chronic wounds are colonized with microbiota, its importance, in the absence of clinical infection, is currently unknown. In this competitive renewal, we hypothesize that host response, wound healing, and clinical DFU outcomes are determined by 1) genomic diversification of the wound pathogen Staphylococcus aureus; 2) commensal interactions with wound pathogens; and 3) commensal interactions with the host. In the previous cycle, we developed a shotgun metagenomic sequencing pipeline to analyze the microbiota colonizing neuropathic, non-infected DFU (n=100; the “DFU100” cohort) in a longitudinal prospective cohort study. We observed that strain-level variation of the wound pathogen Staphylococcus aureus was associated with DFU outcomes. Therefore, in Aim 1, we will use a microbial genomic approach and matched clinical isolates from the DFU100 cohort to identify S. aureus genomic determinants of pathogenesis in DFU and their association with clinical outcomes. We also observed that species clinically regarded as “bystanders” (e.g. skin commensals, environmental contaminants) influence the virulence of wound pathogens and tune host tissue repair responses to promote healing in vivo. Aim 2 will determine if a skin commensal, Corynebacterium striatum, tunes the virulence of S. aureus and improves wound healing in murine and porcine models of S. aureus wound infection. Aim 3 is based on our observation that Alcaligenes faecalis wound isolates promote keratinocyte migration, cytokine secretion, and accelerated wound closure in a murine model of diabetic wound healing. We will establish the mechanism and therapeutic potential of A. faecalis-mediated host responses that lead to accelerated wound healing. The proposed research will use cutting-edge, cross-disciplinary approaches to investigate interactions between wound pathogens, wound “bystanders”, and the host; understanding these mechanisms will lead to improved DFU outcomes as our long-term objectives are to 1) develop novel microbiota-based interventions to improve healing that exploit microbial interactions with each other and the host; and 2) identify microbial biomarkers to classify patients at risk of complication.