Culturing Uncultivatable Gut Microorganisms

培养不可培养的肠道微生物

• 批准号: 8330380
• 负责人: Kim Lewis
• 金额: $ 17.47万
• 依托单位: NORTHEASTERN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-08-01 至 2013-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8330380
• 关键词: Aerobic; Bacteria; Biological Assay; Cells; Coculture Techniques; Collection; Commit; Deposition; Dialysis procedure; Diffusion; Disease; Environment; Feces; Genome; Goals; Growth; Growth Factor; Health; Human; Human Microbiome; In Situ; In Vitro; Individual; Intestines; Laboratories; Lead; Marine Sediment; Marines; Membrane; Methods; Microbe; Microbial Biofilms; Microbiology; Molecular; NIH Program Announcements; Natural Products Chemistry; Organism; Production; Role; Sampling; Sequence Determination; Siderophores; Specialist; Structure; Suspension substance; Suspensions; Taxon; Testing; Universities; Validation; Variant; Washington; Work; base; cell growth; genome sequencing; medical schools; microbiome; microorganism; novel; public health relevance; rRNA Genes; tool

DESCRIPTION (provided by applicant): The majority of gut microbes remain uncultivatable, and this significant obstacle must be overcome to understand the role of the microbiome in human health. The goal of this project is to develop a high-throughput method to grow previously uncultivatable bacteria. Our previous work with uncultivatable microorganisms from the external environment has lead to a number of advances: (1) it is possible to cultivate a substantial number of otherwise uncultivatable bacteria by growing them in situ. When microorganisms are placed into a diffusion chamber which is then returned to their natural environment, a substantial proportion of otherwise uncultivatable microorganisms will grow; (2) reinoculation from chamber to chamber produces domesticated variants that can grow on synthetic media in vitro; (3) many uncultivatable species will grow on synthetic media in co-culture with a cultivable organism from the same environment; (4) we recently discovered the first growth promoting factors for uncultivatable bacteria. An assay-driven purification lead to the identification of siderophores as essential factors produced by helper organisms that trigger growth of uncultivatable bacteria from marine sediment. We find that growth co-culture can be used to obtain uncultivatable organisms from the gut flora. In this project, we will develop a high-throughput approach to co-culture in order to obtain a large collection of previously uncultivatable microorganisms from the gut microbiome. A panel of 24 cultivable gut species representing the main taxonomic groups will be arrayed in a microtiter plate and a platform carrying inserts with a 0.2 5m pore membrane will be placed in the wells. In this manner, each well will be separated into a bottom section inoculated with a given cultivable species and a top section connected with it through pores of the membrane. A suspension from a human fecal sample will then be separated by a cell sorter, and individual cells will be deposited in the upper chamber of each well. After incubation, material from both parts of a well will be collected and tested for growth of the two organisms separately and in co-culture. This will lead to the isolation of uncultivatable species and their helpers. 16S rRNA gene sequence determination will then identify these microorganisms. Whole genome sequencing will be performed for at least ten of the uncultivatable isolates from a variety of taxonomic groups. The genome sequencing will provide an ultimate validation of the proposed approach to obtain novel uncultivatable species from the microbiome. Growth factors will be isolated from the supernatant of corresponding helper organisms by bioassay-guided purification. Structures of the new compounds will be determined. The growth factors will then be examined, individually and in combination, for their ability to enable in vitro cultivation of uncultivatable microorganisms. The tools and approaches we develop are likely to lead to the cultivation of many gut bacteria, and will help us understand the role of the gut microbiome in health and disease. PUBLIC HEALTH RELEVANCE: The majority of gut bacteria are uncultivatable, and do not grow under laboratory conditions for unknown reasons. We find that many of these organisms depend on neighboring, cultivable species for growth. In this project, we will develop a method for large-scale isolation of previously uncultivatable microorganisms by pairing them with the correct helper species, which will enable their genome sequencing, and detailed study of their role in health and disease.

描述(由申请人提供)：大多数肠道微生物仍然无法培养，必须克服这一重大障碍，才能了解微生物群在人类健康中的作用。该项目的目标是开发一种高通量方法来培养以前无法培养的细菌。我们以前对外部环境中不可培养的微生物所做的工作取得了一些进展：(1)通过原位培养，有可能培养出大量原本不可培养的细菌。当微生物被放置到扩散箱中，然后再回到它们的自然环境中，很大一部分原本不可培养的微生物将会生长；(2)从一个舱到另一个舱的重新定位产生了驯化的变体，可以在体外的合成培养基上生长；(3)许多不可培养的物种将在合成培养基上与来自相同环境的可培养生物共培养；(4)我们最近发现了第一个针对不可培养细菌的生长促进因子。化验驱动的纯化导致铁载体被鉴定为辅助生物产生的基本因素，这些辅助生物引发了海洋沉积物中不可培养细菌的生长。我们发现，生长共培养可以用来从肠道菌群中获得不可培养的微生物。在这个项目中，我们将开发一种高通量的共培养方法，以便从肠道微生物群中获得大量以前无法培养的微生物。代表主要分类群的24个可培养肠道物种的小组将排列在微型滴定板中，并将在井中放置一个平台，该平台携带带有0.2 5米孔膜的插件。以这种方式，每口井将被分成接种了给定可栽培物种的底部部分和通过膜的孔与其连接的顶部部分。然后，来自人类粪便样本的悬浮液将被细胞分类器分离，单个细胞将被存放在每口井的上室中。孵化后，将从水井的两个部分收集材料，并分别测试这两种生物的生长情况，以及在共同培养中进行测试。这将导致无法栽培的物种和它们的助手被隔离。16S rRNA基因序列测定将对这些微生物进行鉴定。将对来自不同分类群的至少10个不可培养的分离物进行全基因组测序。基因组测序将最终验证所提出的从微生物组中获得新的不可培养物种的方法。生长因子将通过生物测定引导纯化从相应辅助生物的上清液中分离出来。新化合物的结构将被确定。然后，将分别和组合检查生长因子是否能够在体外培养不可培养的微生物。我们开发的工具和方法可能会导致许多肠道细菌的培养，并将帮助我们了解肠道微生物群在健康和疾病中的作用。 与公共卫生相关：大多数肠道细菌是不可培养的，并且不能在实验室条件下生长，原因不明。我们发现，这些生物中的许多依赖于邻近的可培养物种来生长。在这个项目中，我们将开发一种大规模分离以前无法培养的微生物的方法，方法是将它们与正确的辅助物种配对，这将使它们能够进行基因组测序，并详细研究它们在健康和疾病中的作用。