Multi-method investigation and characterization of the ocular microbiome

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The human body is host to trillions of commensal bacteria, archaea, and fungi whose total gene content is estimated to be over 2000 times more than that of their human hosts, most of which reside in the intestinal tract and colonize many other body sites, notably skin and mouth. The Human Microbiome Project, launched in 2007 by the NIH, has been instrumental in driving the development of multi-omic tools to characterize microbiomes, however the vast majority of studies have focused on studying the microbiome of the large intestine. Moreover, these studies have served to simply sequence these communities, with little effort put forth to cultivate and functionally characterize them or their individual bacterial strains. Therefore, cultivation and characterization of microbial communities at sites less well studied than the gut is a key, unmet need in the field of microbiome research. The ocular surface was initially thought to be sterile due to difficulty in obtaining viable microbes using traditional culture-based techniques, not to mention the potent anti-microbial defenses present in tears. Recent work has indicated the ocular surface is not sterile but contains a low biomass microbial community, which suggests these microbes may play a role in ocular health. However, because most studies characterizing the eye microbiome have focused solely on 16S rDNA and metagenomic sequencing to date, the presence of a bona fide ocular microbiome is still poorly understood. In response to this U24 RFA, we propose to better define the breadth of microbes and microbial communities that populate the ocular surface through a combination of targeted cultivation and metagenomic sequencing approaches. Once individual microbes and communities have been cultivated and cataloged, we will functionally assess the impact of these microbes on various aspects of human eye biology. We have assembled a multidisciplinary team with complementary expertise in ocular biology, immunology, microbial ecology and physiology, metagenomics and microbial cultivation to generate robust resources of microbial strains, genomes, metagenomes, and functional repertoire for the eye and microbiome communities. The following goals to be pursued in generating these resources are: Goal 1: Cultivation and characterization of the ocular surface microbiome. We will use a combination of targeted cultivation, full length (FL) 16S rDNA sequencing, and whole genome shotgun (WGS) metagenomics to characterize the microbiomes associated with the ocular surface including the conjunctiva, tears, and periocular skin. Goal 2: Functional characterization of microbes and microbial community interactions with the eye. To understand how eye associated microbes can impact eye health and physiology, we will combine cultivation of individual microbes from the ocular surface with primary human cell culture assays to monitor the functional impact of microbes on the eye. Our ultimate goal by the end of this proposal period is to have a robust set of well-characterized ocular surface microbes with which the scientific community can now begin functional characterization of eye/microbiome interactions.