Project Summary The growing antibiotic resistance problem requires that we urgently develop and test new approaches to controlling bacterial infections. Phage therapy approaches offer great promise, but significant knowledge gaps currently exist that limit their application. Two issues that currently limit the broader use of phage therapy are the problem of curating specific lytic phage strains for each infection, and the difficulty of delivering phages directly to the infection sites where they are needed. We hypothesize that bacterial pathways that typically promote biofilm-associated growth can be decoupled from the stress- response pathways capable of inducing resident prophages. By separating these cellular responses, we propose to stabilize biofilms while also inducing temperate phages already present in the bacterial genome to enter lytic phase and kill the host cell. In so doing, we effectively bypass the limitations imposed by finding and delivering strain matched lytic phages to infections, while also developing approaches that make use of temperate phage therapy approaches. In Aim 1, we propose to determine biofilm dispersal rates and ascertain how biofilm-associated growth can be manipulated using cellular pathways. In Aim 2, we will analyze mechanisms by which lysogens can be induced into lytic phase to kill their hosts without spreading virulence factors beyond the biofilm. In Aim 3, we reinforce the approaches of the previous aims with an optimized lytic phage ambush of any escaping dispersers. The goals of this proposal are to develop an innovative, multi-pronged approach to phage therapy for biofilm-associated bacteria while also increasing overall knowledge of the limits and applicability of phage therapy.