The role of colonizing and infecting pathogens (wound bioburden) on diabetic foot ulcer (DFU) outcomes remains unclear. Our group has shown that the totality of microorganisms in DFUs measured using high throughput 16S ribosomal RNA (rRNA) gene sequencing, is directly related to: ulcer duration, ulcer depth, and poor glycemic control. However, changes in bioburden over time provided only limited insights into the role of these changes on DFU outcomes. Nonetheless, bioburden may be influencing DFU outcomes, but the specific dimensions of importance may not be discernable using 16S rRNA profiling. Major limitations of this molecular technique include the fact that it does not disclose functional potential of the microbiota and resolution to species or strain level is often impossible. To circumvent these limitations, we will employ whole genome shotgun (WGS) metagenomic sequencing and analysis, a powerful lens for deciphering the functional potential of microbial communities. Our overarching hypothesis is that specific dimensions of the dynamic DFU microbiome are predictive of outcome. Additionally, we hypothesize that different treatment regimens have different effects on the DFU microbiome. We have assembled a multi-disciplinary team of experts to execute the aims of this proposal, with basic science expertise in wound microbiome and metagenomics and clinical and research expertise in DFUs and infection. To test our hypothesis, we will complete the following aims: Aim 1: Determine if pathogenic species/strains and overall microbial diversity of the DFU metagenome are associated with specific DFU outcomes. We will characterize the changes in metagenomes over time for 107 subjects with DFUs recruited from our previous studies, from whom specimens were collected every 2 weeks from presentation to final outcome. We will determine if 1) the presence of specific species or strains of pathogens (e.g. beta-hemolytic Streptococcus); and 2) low microbial diversity are directly associated with lower rates of healing or infection-related complications (i.e., ulcer deterioration, osteomyelitis or amputation). Aim 2: Determine if pathogenicity genes in the DFU metagenome are associated with specific DFU outcomes. We will examine the presence and relative abundance of genes conferring 1) biofilm-formation potential, 2) antibiotic resistance and, 3) virulence factors, to determine if they are association with lower rates of healing and infection-related complications. Aim 3: Determine the effects of aggressive sharp debridement and antibiotic treatment on the DFU metagenome. This is the first longitudinal study of the role of the DFU microbiome on DFU outcomes using WGS metagenomic sequencing as well as the first to examine the impact of two commonly employed antimicrobial interventions on the DFU metagenome.