Project Summary The human mucosal surface is a complex ecosystem made of bacteria, viruses, epithelial cells, mucus, and molecules such as proteins, sugars, and other solutes whose balance is key to the health of the host. It is also the first line of defense against invading bacteria, and a site of colonization by diverse microbiota. Some members of this microbiota, termed pathobionts, cause serious local and systemic infections. However, the use of antibiotics to treat these infections is a classic Catch-22; there may temporary relief but down the road the very solution generates a bigger version of the original problem. Whereas one may rid the environment of the pathobiont, one also eliminates the beneficial microbiota that antagonize the pathobiont while creating a selection that further increases rates of resistance. In addition, the destruction of the balance of the ecosystem further predisposes that system to invasion by other pathobionts, and, in the long run, increases the risk of infection and inflammation. There is a real need to develop an alternative line of antibacterials that lack these limitations. The overall objective of this project is to discover bacteriophage, viruses that infect and kill bacteria, that are specifically active against drug-resistant pathobionts in the complex environment of a human mucosal surface. These phage should be specific for their bacterial target, as well as have evolved features that promote enhanced activity in the face of the complexity presented by such surfaces. Furthermore, should the pathobiont spread systemically, these phage should synergize with conventional antibiotics while simultaneously generating a steep evolutionary path for the emergence of new resistance. Using one of the world’s largest collections of therapeutic phages and characterizing them for enhanced activity in human mucosal biomimetics, the research program described here lays the foundation for the development of a novel class of mucosal-active antibacterials that clear problematic pathobionts while simultaneously maintaining balance of the native microbiota.