This proposal is based on a long-standing and intriguing clinical observation that is directly relevant to human health and concerns the dynamics of human nose and throat microbial communities: about 50% of individuals are not colonized with either Staphylococcus aureus or Streptococcus pneumoniae, and are consequently at low risk for infection by these important pathogens. We hypothesize that these individuals harbor mutualistic antibiotic-producing bacteria in their nose and throat that selectively inhibit the colonization and proliferation of S. aureus and/or S. pneumoniae. Our recent work suggests that in the nostrils, commensal Actinobacteria of the genera Corynebacterium and Propionibacterium inhibit colonization by S. aureus. We have identified strains of these colonization-blocking bacteria that produce small molecules that inhibit S. aureus growth. These beneficial microbes-and the antibiotics they produce-could serve as the basis for novel small molecule and probiotic therapies. An understanding of the role of small-molecule-mediated interactions in the dynamics of nose and throat microbial communities will allow us to develop strategies for preventing and/or eliminating pathogen carriage, drastically decreasing the risk for infection. To test this hypothesis, we take advantage of a unique set of microbiota samples already collected as part of an NIH-funded study of S. pneumoniae carriage. This unique and valuable sample set captures a stage of human development when carriage of S. aureus and S. pneumoniae is in flux; S. pneumoniae carriage rates peak at ~45% in toddlers, a time when S. aureus carriage rates are at a low of ~10%, then S. aureus carriage rates rise as children approach school age reaching ~30% by adolescence. We will use a multi-disciplinary strategy broken down into three aims. In Aim 1, we will use deep sequencing of an extant set of 200 paired nostril and nasopharyngeal microbiota samples to determine which taxa are inversely related with culture-proven S. aureus and S. pneumoniae, and co-culture assays to identify which of these candidate colonization-blocking bacteria produce an antibiotic. In Aim 2, we will use bioassay-guided fractionation to purify these antibiotics from Aim 1 and NMR and MS to solve the their structures. In Aim 3, we will use a combination of bioinformatics and bacterial genetics to identify the genes that encode these antibiotics and we will test whether the presence of antibiotic-encoding genes is negatively correlated with the presence of S. aureus and S. pneumoniae in community metagenomic data from Human Microbiome Project samples. Our efforts will identify new antibiotics that modulate nose and throat microbial community dynamics and impact colonization and infection by two important bacterial pathogens: S. aureus and S. pneumoniae.