Project Summary Before the discovery of antibiotics, infectious diseases were the three leading causes of death in the United States and constituted nearly 50% of the deaths annually. Today only one infectious disease, pneumonia, is among the top 10 causes of death in the US, largely due to the success of antibiotics in treating bacterial infections. Unfortunately, the public health benefits provided by antibiotics are at serious risk due to the emergence of antibiotic resistant bacteria, an inevitable consequence of the evolutionary pressure exerted on bacteria by these drugs. Eventually the bacterial pathogens we hope to keep at bay will become resistant to all clinically relevant antibiotics, plunging humans back into a pre-antibiotic world in which infectious disease is the leading cause of death. Therefore, we need to find innovative, and rapidly implementable, ways to reduce or supplement antibiotics to preserve their utility in controlling bacterial infections. Many bacterial pathogens, termed pathobionts, reside within the human microbiota in the absence of disease and only instigate pathogenesis after disruption of the microbial community driven by abrupt environmental changes such as acute inflammation. While there is general acceptance that the commensal microbes provide pathogen colonization resistance and suppression of pathobiont virulence in a healthy state, the mechanistic understanding for how they provide these benefits is lacking. In this project, we explore using the human microbiome to identify ecological principles that allow for the design and implementation of microbial communities that suppress bacterial pathogens. We have selected Clostridioides difficile and extraintestinal pathogenic E. coli (ExPEC) as the two main pathogens to study as they are deemed antibiotic resistance threats by the CDC and necessitate millions of antibiotic prescriptions each year. Even with antibiotic treatment, recurrent infections with both of these pathogens is common, and there is currently a lack of long-lasting preventative strategies. Using a novel method to simplify human microbiome communities and advanced in vitro human tissue culture and humanized murine models, we seek to identify key microbial consortia for suppressing these pathogens. We ultimately expect to optimize a small number of defined microbial communities that can be used to eradicate or prevent these infections in people.