Defining bacterial members of the ocular surface microbiome and assessing stability over time

项目总结 最近的研究表明，眼部微生物群存在并影响眼表。 健康和疾病。尽管如此，眼表微生物组的“核心”成分一直是 困难很大程度上是因为眼睛的生物量低，缺乏可靠的数据集，只能依赖NEXT 世代测序(NGS)，以及无法显示微生物和宿主之间的因果关系 生理学。在此，我们概述了一项直接解决这些知识差距的提案，并将 为未来的研究奠定基础，旨在调查眼表微生物群如何影响健康和 疾病。我们招募了一支由临床和基础领域的专家组成的多学科研究团队 眼科研究、低生物量部位的微生物组和免疫学。有了这个团队，我们计划首先 描述两个地理位置(匹兹堡和迈阿密)的健康人眼微生物群 纵向(0、1周、3个月)使用分子技术(DNA和RNA测序)和培养法。 虽然分子技术将为我们提供最广泛的细菌联合体，但培养物学将为我们提供 我们计划在坎贝尔实验室储存有活性的细菌，这样来自健康人眼的细菌 可以在未来的研究中为社区提供资源。接下来，因为免疫力通常产生于 长期与宿主保持联系的微生物组分，我们计划使用 人类针对眼部细菌的免疫反应以区分定植细菌和暂时性细菌 可能被冲走的细菌。为了进一步加深对眼部定植细菌的了解，我们计划 用人眼部细菌接种无菌(GF)和无特定病原体(SPF)小鼠。在延长了 在一段时间内，我们计划从小鼠的眼睛中重新分离细菌。仍然存在于眼睛上的细菌 表面将被认为具有更高的可能性，在人类的眼睛表面殖民者。这个 开发这样的模型将允许其他人评估其他细菌的定植能力，这些细菌可能 被认为是眼表微生物群的核心组成部分。总而言之，我们提案中的数据将是 为整个社区提供资源，因为我们将拥有：1)生成一组强大的眼部微生物群 将提供关于眼微生物组特征的稳定性和一致性的信息的测序数据， 2)创建了一个从健康人眼获取的细菌库，3)定义了使用人类 免疫来衡量细菌的定植能力，4)开发了一个活体动物模型来评估 眼睛细菌可以接种，并从眼睛中重新分离出来。这些资源将免费用于 并将作为进一步研究眼部微生物群如何影响眼表面的基础 健康和疾病。

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.