Project Summary/Abstract Multidrug-resistant bacteria are an increasing health threat. Most new antibacterial agents are natural products that are made by bacteria, or their synthetic derivatives. This is because, over evolutionary time, bacteria gain the ability to synthesize small molecules that very selectively target essential processes in other bacteria. One problem in identifying good antibacterial agents that are relatively nontoxic to people is that early drug discovery assays cannot determine whether compounds are truly selective until well into the drug development process. There is no reason that randomly cultivated bacteria from the environment would produce chemicals that are benign in humans, yet lethal to competing bacteria. Finding those compounds, or improving existing compounds to minimize toxicity and improve antibacterial activity, requires a significant investment. Here, we propose to examine symbiotic bacteria that are compatible with animal physiology, and for which evolution favors the production of selective antibacterial agents. The bacteria are enriched within animal hosts, where they produce hundreds or perhaps thousands of small molecule natural products. One of the main ecological roles of the symbiotic bacteria is likely to clear other bacteria from the host. Proof-of-concept experiments demonstrate that the bacteria produce nontoxic compounds that very selectively target human pathogens, including some of the most lethal bacterial pathogens that infect humans. In this program, we will optimize identified, chemically novel antibacterial compounds by understanding their mechanisms of action and by investigating and improving their existing properties further through chemical synthesis. Further, we will continue to identify and characterize promising new antibacterial agents that are previously unknown to science. Our long-term goal is to provide a novel pipeline of new antibacterial agents to combat multidrug resistance.