Decades of overuse have allowed bacteria to evolve resistance against nearly every existing antibiotic. This has resulted in increased mortality and hospital costs associated with bacterial infections. There is a dire need to develop novel strategies to control bacterial growth that reduces the evolution of resistance. As cooperation is required by many bacteria to cause infections and to resist antibiotics, disrupting cooperation is an attractive mechanism to control bacterial growth. One overlooked mechanism to disrupt cooperation is to physically disrupt the spatial structure of a bacterial population. Accordingly, the objective of this proposal is to determine how periodic spatial disturbances caused by physical force affects cooperation in bacteria. The central hypothesis of the proposed research is that periodic spatial disturbance will cause a spatiotemporal mismatch of signaling molecules called autoinducers (AI) and bacteria, leading to disruption of cooperation between bacteria. Synthesis and accumulation of a diffusible AI is required for many bacteria to cooperate. Most of the AI is not retained by the bacterium that produces it. Therefore, the concentration of the AI detected by each bacterium will be dictated by the spatial distribution of bacteria. As the movement of bacteria and AI occurs on different timescales during a disturbance, this will decouple their spatial positioning, and affect the ability of bacteria to cooperate. The rationale for the proposed research is that once an understanding of how periodic spatial disturbance can affect cooperation has been developed, periodic disturbance of bacterial populations can be used to reduce bacterial cooperation and growth, which will lead to novel mechanisms to reduce the growth and spread of bacterial infections. Based on preliminary data, we proposed two aims: 1) determine how periodic spatial disturbance affects cooperation in Pseudomonas aeruginosa and 2) develop a mathematical framework to explore how cooperation is perturbed in biofilms. In Aim 1, we will use molecular biology techniques, microscopy and mathematical modeling to determine how periodic spatial disturbance affects cooperation by measuring virulence factors and gene expression. In Aim 2, we will create a 3D agent-based model that captures how periodic spatial disturbance affects cooperation in a biofilm. Our research is innovative as it uses a physical force to disrupt cooperation, which may be less prone to driving the evolution of resistance. Our research is significant, as it will lead to a mechanistic understanding of how periodic disturbance of bacteria affects cooperation. This may lead to novel strategies to reduce bacterial infections, which will reduce our use of antibiotics.