Abstract Skin and soft tissue infection (SSTI) is a major complication in diabetic patients and Staphylococcus aureus is the most common causative organism. Antibiotics frequently fail to clear these infections, leading to chronic infection and progression to more severe infections such osteomyelitis and bacteremia. The reasons for the high rates of treatment failure in diabetic patients remain unclear. We employ a murine SSTI model with normal and diabetic mice and methicillin-resistant Staphylococcus aureus (MRSA). We observe increased antibiotic tolerance and spontaneous antibiotic resistance (mutation) in diabetic mice infected with MRSA, compared to the infected normal mice. We also observe a 10-fold increase in glucose concentrations in the diabetic infection environment. We hypothesize that excess glucose in the diabetic infection environment alters bacterial and host metabolism driving antibiotic tolerance and resistance. In aim 1 we will examine how excess glucose primes glycolysis in S. aureus, leading to acidification of the infection microenvironment and increased mutagenesis, resulting in antibiotic tolerance and resistance. In aim 2 we will examine how incapacitation of the immune system in diabetic mice may be inducing reservoirs of antibiotic tolerant and resistant S. aureus during infection. In aim 3, we will examine the in-host evolution of antibiotic tolerance, resistance, and fitness during sequential infection of diabetic mice to determine the progression of mutations that result in highly virulent, antibiotic resistant strains that are likely highly deleterious to the patient. Determining how blood glucose levels contribute to the development of antibiotic resistance will be an important development and will further emphasize the importance of treating and preventing diabetes, particularly as rates continue to rise annually.