PROJECT SUMMARY/ABSTRACT The global spread of antibiotic resistance is producing a human health crisis. The objective of this application is to investigate a novel combination therapy, by using antimicrobial blue light (aBL) with antibiotics, for synergistic antimicrobial activity and/or for the preservation/potentiation of the potency of conventional antibiotics. Our central hypothesis is that aBL+antibiotics will provide a broader action spectrum and improved antimicrobial activity through a multifunctional mode of action with both synergistic and adjuvant capabilities. To address this hypothesis, we have formulated three Specific Aims. In Aim 1, we will first investigate the potential for aBL to synergize with antibiotics against bacteria in vitro. We will next examine whether aBL can mitigate or reverse bacterial β-lactam resistance, repurpose glycopeptide and penicillin antibiotics for application against Gram (-) bacteria, and mitigate or reverse bacterial efflux- mediated resistance. For the safety study, we will evaluate the effects of aBL+antibiotics on human cell viability, inflammatory response, oxidative stress, DNA damage, and functions of human cells. Additionally, we will determine whether bacteria can develop resistance to aBL+antibiotics. In Aim 2, we will elucidate the mechanism of action of aBL+antibiotics. To this end, we will first analyze the individual contributions of different reactive oxygen species (ROS) to the overall ROS upregulation induced by aBL+antibiotics in bacteria. We will next assess the effects of aBL on bacterial β-lactamase activity and expression, the outer membrane permeability of Gram (-) bacteria, and bacterial efflux activity and expression. Finally, we will examine potential changes to bacterial transcriptome following aBL+antibiotics. In Aim 3, we will determine the efficacy of aBL+antibiotics in vivo for treating cutaneous abscesses in mice infected with methicillin-resistant Staphylococcus aureus and tympanostomy tube otorrhea (TTO) in chinchillas infected with Pseudomonas aeruginosa. To treat cutaneous abscesses, we will utilize a novel dual-functional microneedle array to deliver aBL and levofloxacin simultaneously and interstitially to the infection sites. To treat TTO, we will use our newly invented optical tympanostomy tube (Optical-TT), which emits aBL uniformly from its entire tube surface, in conjunction with sulfamethoxazole ear drops, to eradicate biofilms on the tube surface. Additionally, for the safety study, we will first determine the viability loss of the host cells in animals following aBL+antibiotic treatment. We will also evaluate the effects of aBL+antibiotics on wound healing and inflammatory response in animals. Finally, we will determine whether there is a long-term carcinogenic effect associated with aBL+antibiotics. If the strategy of aBL+antibiotic combinations can be shown to be both effective and safe, it may provide an innovative broad-spectrum regimen to combat anti...