Antimicrobial resistant (AMR) Enterobacteriaceae are a leading cause of worldwide mortality. Severe infection results when species of this family translocate from the gastrointestinal tract to the bloodstream. The research proposed herein aims to identify the genes and gene networks in Enterobacteriaceae that drive translocation, characterize how the human host epithelium responds, and determine if the commensal microbiota can counteract this process. In the first aim, hundreds of clinical strains will be sequenced, annotated, and examined for their ability to transverse the epithelial mucosal barrier in a human organoid model of infection. The empirically derived data will be used to train a machine learning approach to infer virulence potential for genomic data alone. Predictions will then be tested and validated in the organoid model. In the second aim, the complete host transcriptional response to translocators and non-translocators will be mapped across several organoid lines. Commensal species that antagonize translocators will be discovered and assessed for synergy with host defenses to define the collective host-biota nexus that prevents disease. The project will yield new vaccine targets against AMR bacteria, human risk-factors associated with severe infection, and therapeutic probiotics that block translocation.