PROJECT SUMMARY/ABSTRACT Staphylococcus aureus is a leading cause of infections worldwide. In recent decades, the treatment of S. aureus infections has become increasingly difficult because of the increasing incidence of antibiotic resistance, justifying the need for developing alternative therapeutics. Antimicrobial blue light (aBL; 405 nm wavelength), an innovative nonpharmacological approach, has attracted increasing attention due to its intrinsic activity against a wide range of bacteria irrespective of their antibiotic resistance profiles. However, it has also been found that S. aureus is much more tolerant of 405 nm aBL than most other bacteria. A potential reason for the higher tolerance of S. aureus to aBL is the presence of staphyloxanthin (STX) in S. aureus. STX acts as an antioxidant and facilitates the tolerance of S. aureus to oxidative stress generated by 405 nm aBL. Fortunately, a recent study discovered that STX is prone to photobleaching by 460 nm wavelength irradiation. This finding leads to our central hypothesis that 460 nm irradiation would potentiate the susceptibility of S. aureus to killing by 405 nm aBL. To test this hypothesis, we propose two Specific Aims: In Aim 1, we will first investigate the efficacy of the dual-wavelength irradiation (DWI; 460 nm followed by 405 nm) strategy for killing S. aureus in vitro. A panel of S. aureus isolates, including ATCC reference strains and recent clinical isolates, will be selected. Both planktonic bacteria and biofilms will be studied. We will also assess the effect of the pre-application of 460 nm irradiation on the 405 nm aBL-induced production of reactive oxygen species in S. aureus. Additionally, as the safety study, we will evaluate the cytotoxicity and genotoxicity of DWI to normal human cells by treating human cells under therapeutic exposures of DWI for eradicating S. aureus. Finally, we will determine whether DWI affects the ROS-producing capability of the immune cells. In Aim 2, we will conduct a preclinical study to determine the efficacy and safety of DWI for treating cutaneous S. aureus abscesses in mice. We will use a bioluminescent strain of methicillin-resistant S. aureus (MRSA USA 300) to infect mice, thus allowing real-time monitoring of the extent of infection in living animals using bioluminescence imaging. DWI will be initiated at varying time points (30 min, 4 h, or 48 h) after infection. To facilitate light penetration in biological tissues, we will use a novel optical lens-microneedle array patch to deliver light interstitially to the infection sites. The efficacy found with DWI will be compared with that of systemic tetracycline, an empirical antibiotic therapy in clinic for cutaneous S. aureus infections. As the safety study, we will determine the effects of DWI on the viability and DNA damage of the host cells, wound healing, and inflammatory response in treated tissues. Collectively, the successful completion of the Specific Aims outlined in this applic...