Project 3 The overall objective and significance of Project 3 is to generate preclinical validation of nasal microbiota-based therapeutics to block nasal colonization by Staphylococcus aureus and Streptococcus pneumoniae. Our innovative approach to achieve this is to cultivate human nasal bacteria on human nasal organoids. Together, S. aureus and S. pneumoniae account for 39% of cases and 29% of deaths attributed to antibiotic-resistant bacteria in the U.S. Nasal colonization is the primary source of invasive infection by each and the reservoir for their transmission; blocking colonization reduces both infection and transmission. Lacking effective vaccines against S. aureus or nonvaccine serotypes of S. pneumoniae, there is an urgent need for new nonantibiotic approaches to prevent infections by both. Current therapies for nasal decolonization of S. aureus depend on a topical antibiotic and are temporally correlated with rising antibiotic resistance. Here, we address a gap in knowledge in how to repopulate with pathobiont-resistant microbiota to improve long-term outcomes. Multiple nasal microbiota studies report benign bacterial species present when adults are free of S. aureus and when children are free of S. pneumoniae. For example, nasal colonization by the benign bacterium Dolosigranulum pigrum, often with harmless Corynebacterium, is associated with the absence of S. pneumoniae in children. In adults, D. pigrum is inversely associated with S. aureus nasal colonization. Our preliminary data indicate many strains of D. pigrum inhibit S. aureus growth in vitro. Also, cocultivation of D. pigrum with Corynebacterium pseudodiphtheriticum robustly inhibits S. pneumoniae growth in vitro, versus either alone. Moreover, Corynebacterium enhance D. pigrum growth. Thus, our overarching hypothesis is that reproducible consortia of benign human nasal bacteria can confer colonization resistance to S. aureus and S. pneumoniae, preventing infection by and transmission of these antibiotic-resistant threats. Moreover, antipathobiont mucosal-active lytic phage may augment this. Networks of interactions underpin microbiota composition. Relying on consortia avoids the risk of unexpected community interactions that can occur when adding a single strain and increases the likelihood of successful repopulation with a desired community. A major hurdle to clinical trials is to identify fully defined consortia of benign nasal bacterial strains that confer colonization resistance in the context of human nasal epithelium and promote a healthy, intact epithelial barrier. Our Organoid Cultivation Core has successfully generated human airway organoids. Thus, we will overcome this hurdle using advances in nasal epithelial organoids combined with our expertise in cultivating human nasal bacteria and identifying molecular mechanisms of interactions within nasal microbiota. With methods routinely used by us, Project 1 and the Organoid Cultivation Core, we will achieve two aims: identify and characterize reproducible human nasal microbial consortia to 1) protect nasal epithelium from colonization by S. aureus/S. pneumoniae and 2) to promote epithelial barrier integrity and block epithelial invasion by S. aureus/S. pneumoniae in vitro, and identify underlying mechanisms.
