PROJECT SUMMARY Recent studies have shown data suggesting that the ocular microbiome exists and influences ocular surface health and disease. Despite this defining the “core” components of the ocular surface microbiome has been difficult largely due to the low biomass nature of the eye, a lack of robust data sets, reliance only on next generation sequencing (NGS), and the inability to show causal relationships between microbes and host physiology. Herein, we have outlined a proposal that directly addresses these knowledge gaps and that will lay the foundation for future studies aimed at investigating how the ocular surface microbiome influences health and disease. We have recruited a multidisciplinary research team that consists of experts in clinical and basic ophthalmology research, microbiome of low biomass sites, and immunology. With this team, we plan to first characterize the healthy human ocular microbiome in two geographic locations (Pittsburgh and Miami) longitudinally (0, 1 week, 3 months) using molecular techniques (DNA and RNA sequencing) and culturomics. While molecular techniques will provide us with the broadest consortia of bacteria, culturomics will provide us with viable bacteria that we plan to bank in the Campbell Laboratory, so that bacteria from healthy human eyes can be a resource for the community in future studies. Next, because immunity is normally generated towards components of the microbiome that remain associated with the host for extended periods of time, we plan to use human immune responses directed against ocular bacteria to distinguish colonizing bacteria from transient bacteria that are likely washed away. To further refine the understanding of ocular colonizing bacteria, we plan to inoculate germ free (GF) and specific pathogen free (SPF) mice with human ocular bacteria. After an extended period of time, we plan to re-isolate bacteria from the eyes of mice. Bacteria that are still present on the ocular surface will be considered to have a higher likelihood of being an ocular surface colonizer in humans. The development of a model such as this will allow others to assess the colonizing ability of other bacteria that may be considered a core component of the ocular surface microbiome. In sum, data from our proposal will be a resource for the community at large because we will have: 1) generated a robust set of ocular microbiome sequencing data that will provide information on the stability and consistency of ocular microbiome signatures, 2) created a bank of bacteria that were acquired from healthy human eyes, 3) defined protocols to use human immunity to measure the colonizing ability of bacteria, 4) developed an in vivo animal model to assess how ocular bacteria can be inoculated and re-isolated from the eye. These resources will be free to use for the community and will act as a base to further investigate how the ocular microbiome influences ocular surface health and disease.