Abstract: The discovery of antibiotics in the early 20th century has transformed modern medicine; yet decades of use, overuse, and misuse have culminated in the rapid rise in pathogens that are refractory to our existing drugs. Antibiotic resistance is not the only reason for treatment failure. Within antibiotic-sensitive cultures, subpopulations of bacteria can transiently reprogram their phenotype, which enable them to survive lethal antibiotic doses. These bacterial persisters are thought to underlie recurrent and chronic infections, and they can fuel the development of resistance. Mounting evidence shows that environmental factors modulate phenotypic changes that lead to antibiotic persistence before, during, and after treatment. As such, achieving a deeper understanding of the interplay between environmental cues, bacterial phenotypic responses, and antibiotic susceptibility will improve our ability to devise more effective treatment regimens. When pathogens colonize and infect different parts of the host, they are often exposed to other pathogens and constituents of the host microbiome. The extent to which microbial interactions modulate a pathogen's phenotype and antibiotic persistence remains largely unexplored. Our overarching objective for this project is to systematically investigate the impact of microbiome constituents on Staphylococcus aureus's phenotypic response and persistence to antibiotics. To achieve this goal, we will develop a co-culturing phenotypic screen to identify bacterial strains and communities that impact S. aureus antibiotic persistence toward distinct classes of antibiotics. Using a combination of transcriptomics, metabolomics, and single-cell approaches, we will determine how these microbial interactions modulate S. aureus phenotypes (on the population- and single- cell level) before, during, and after antibiotic treatment. We will also use analytical techniques to identify molecular determinants that mediate microbial interactions responsible for the strongest effect on S. aureus persistence. We envision that the outcome of this project will expand our knowledge of the persister phenotype, contribute to the discovery of novel antimicrobial adjuvants, and guide the development of innovative treatment strategies to tackle chronic infections.