The microbial basis of impaired wound healing: differential roles for pathogens, \"bystanders\", and strain-level diversification in clinical outcomes

Chronic, non-healing wounds are a major complication of diabetes associated with high morbidity and health care expenditures estimated at $9-13 billion annually in the US. Though microbial infection and critical colonization is hypothesized to impair healing and contribute to severe outcomes such as amputation, antimicrobial therapy is inefficacious and the role of microbes in tissue repair, regeneration, and healing remains unclear. Here, in a longitudinal prospective cohort study of 100 subjects with non-infected neuropathic diabetic foot ulcer (DFU), we performed metagenomic shotgun sequencing to elucidate microbial temporal dynamics at strain-level resolution, to investigate pathogenicity and virulence of the DFU microbiome with respect to outcomes, and to determine the influence of therapeutic intervention on the DFU microbiota. Slow healing DFUs were associated with signatures of biofilm formation, host invasion, and virulence. Though antibiotic resistance was widespread at the genetic level, debridement, rather than antibiotic treatment, significantly shifted the DFU microbiome in patients with more favorable outcomes. Primary clinical isolates of S. aureus, C. striatum, and A. faecalis induced differential biological responses in keratinocytes and in a murine model of diabetic wound healing, with the S. aureus strain associated with non-healing wounds eliciting the most severe phenotype. Together these findings implicate strain-level diversification of the wound pathogen S. aureus in chronic wound outcomes, while revealing potential contributions from skin commensals and other previously underappreciated constituents of the wound microbiota.