PROJECT SUMMARY/ABSTRACT This application is for an RO1 project focused on defining determinants of bacterial persistence in biofilms during otitis media infections. Otitis media is caused by airway opportunists such as Haemophilus influenzae (Hi), which forms multicellular biofilm communities that promote bacterial persistence within the middle-ear chamber. Over the past 15 years we have defined mechanism(s) involved in biofilm formation by Hi, proved that biofilms promote bacterial persistence in vivo, and defined metabolic determinants of Hi persistence within biofilm communities. Now, we plan to use this information to better understand the metabolic state of Hi bacteria within biofilms, and to use that information to identify enzymes important to glutathione mediated redox homeostasis to eradicate Hi bacteria within biofilms. In particular, we will ask how glutathione contributes to Hi persistence in the face of chronic oxidative stress. We will complete the following Specific Aims: Specific Aim 1. To define role(s) for glutathione uptake and metabolism in Hi survival in biofilms. Specific Aim 2. To test role(s) for glutathione mediated redox homeostasis in Hi persistence in vivo. Although it is clear that the biofilm mode of growth is involved in the majority of persistent infections, much remains to be learned about the determinants of bacterial resistance to clearance within biofilms. The completion of the proposed work will enhance our understanding of the role of biofilms within the clinical context of otitis media.