The human intestine is an ecosystem of microbes. This ecosystem helps shape immune function, provides scarce nutrients, aids in digestion, and when balanced, maintains health. Amongst the many bacteria that colonize this organ, some harbor virulence factors and carry drug-resistance, the so-called pathobionts, which may be asymptomatic members of the intestinal microbiota. Under certain conditions, mostly related to a loss of immune function, these organisms can cause systemic bloodstream infections that can lead to sepsis or sepsis-like states. As such, they are leading causes of community and nosocomial infections. The most prominent of these are members of the superfamily of Enterobacteriaceae and Enterococcaceae. The most concerning pathobiont members of the Enterobacteriaceae are the Gram-negatives Eschericia coli and Klebsiella pneumoniae, while those of the Enterococcaceae include the Gram- positive Enterococcus faecalis and Enterocccus faecium. There are no vaccines for these species. The focus of this project is to generate the world's first fully annotated and searchable reference library of pathogenic and drug-resistant pathobionts that colonize the human intestine. This library will then be the source of intra-species competition in organotypic models of the human intestine to select for the most fit binders and colonizers of the apical mucosa. These strains then be assessed for their ability to breach the mucosa (translocation), induce global gene expression across a hundred different human lines, and be out-competed or antagonized by members of a small and large intestine commensal community. At the completion of the project, there will be numerous bacterial, host, and commensal targets for new drug development.