Complicated skin and skin structure infections (cSSSIs) including abscesses, burn infections, cellulitis and diabetic foot infections represent an enormous burden on the healthcare industry. Treatment options are limited, and treatment failure is common, frequently leading to chronic wound infection. Staphylococcus aureus is the most common cause of these infections, followed by Enterococcus faecalis. Vancomycin, a lipid II targeting antibiotic that inhibits cell wall synthesis, is essential in the treatment of cSSSI. However, vancomycin-intermediate resistant S. aureus (VISA) and vancomycin-resistant Enterococci (VRE) are a growing problem. Additionally, even in susceptible populations, antibiotic tolerant persister cells can survive vancomycin exposure, making eradication of infection difficult to achieve. Identifying ways to increase the efficacy of vancomycin, to target persister cells and overcome resistance would greatly improve the treatment of cSSSIs. We find that palmitoleic acid, a non-toxic unsaturated fatty acid found in human serum potentiates vancomycin efficacy against S. aureus and E. faecalis. Strikingly, palmitoleic acid also rapidly sensitizes persister cells and resistant populations to vancomycin. Vancomycin causes accumulation of the hydrophobic lipid II molecule at the septum. Our preliminary data suggests that palmitoleic acid is recruited to these hydrophobic regions which destabilize the membrane and cause cell death. Vancomycin-resistant isolates induce expression of resistance genes when they encounter vancomycin. We hypothesize that the rapidity of palmitoleic acid- vancomycin killing may outpace the induction of resistance gene expression, resulting in death of the resistant strains. In this proposal, we will 1) determine how palmitoleic acid/vancomycin kills persister cells and overcomes vancomycin-resistance and 2) evaluate the efficacy of this therapeutic combination in a pre-clinical diabetic wound infection model. Utilizing host-produced unsaturated fatty acids to potentiate vancomycin killing against resistant organisms is conceptually innovative. If successful, this proposal will represent the first steps toward the development of a powerful therapeutic combination for the eradication of recalcitrant gram-positive wound infection.