Cultivating the 'uncultivable' oral microflora

描述(申请人提供)：口腔中有700多种细菌，但其中一半以上无法在体外培养，无法进行研究。这项研究的主要目标是获得“无法培养的”失踪物种。AIM 1利用一种全新的方法在自然环境中种植“不可耕种的作物”(Kaeberlein，T.，Lewis，K.和Epstein，S.S.，2002)。使用模拟自然环境(Science 296：1127-1129)结合将生长设备微型化到纳米级，在纯培养中分离“不可培养”的微生物。纳米室将接种目标微生物的单细胞，并进行原位培养。通过限制细胞移动的薄膜将纳米腔与环境隔开，但允许扩散。这将为细胞提供其自然环境的一整套生长组件。这绕过了标准栽培技术的限制，并将允许在纯培养中生长和分离目前未培育的菌株。在目标2中，我们将重点关注与口腔疾病有关的物种，并将这些物种驯化以在标准的培养皿中生长。我们之前已经成功地从环境样本中驯化了未培养的细菌。该项目的主要成果将是(1)培养“不可培养”的人类口腔微生物群的新方法，以及(2)收集对人类健康具有潜在重要性的新物种。新培育的物种可以很容易地表征，即生物化学和遗传(包括全基因组测序)。我们将把这个收藏提供给其他研究人员，并将创建一个免费的基于网络的数据库来表征这个收藏。这些目标将在三个学术实验室、一名临床医生和一名工业合作伙伴之间的合作下实现。这个跨越五个不同领域的多学科项目将使我们能够实现应用的主要目标：获得具有临床重要性的人类口腔微生物群成员。 不可培养的细菌占人类口腔微生物区系的一半以上，其中可能会发现新的病原体或共生体。我们将开发一种新的方法来分离和鉴定以前不可培养的纯培养口腔细菌。这将导致识别以前未知的影响人类健康的微生物，并将为开发针对口腔病原体的改进疗法奠定基础。

DESCRIPTION (provided by applicant): There are over 700 bacterial species in the oral cavity but over half of these cannot be cultivated in vitro and remain inaccessible for study. The principal goal of this research is to gain access to the "uncultivable" missing species. Aim 1 utilizes a fundamentally new approach to grow "uncultivables" in their natural environment (Kaeberlein, T., Lewis, K., and Epstein, S.S. 2002. Isolating "uncultivable" microorganisms in pure culture using a simulated natural environment (Science 296:1127-1129) combined with miniaturizing the growth devices to nanoscale. The nanochambers will be inoculated with single cells of target microorganisms and incubated in situ. The nanochambers are separated from the environment by membranes restricting movement of cells, but allowing for diffusion. This will provide the cells with the complete suite of growth components of their natural environment. This bypasses the limitations of standard cultivation techniques, and will allow growth and isolation in pure culture of presently uncultivated strains. In Aim 2, we will focus on species implicated in oral diseases, and domesticate these species for growth in a standard Petri dish. We have previously successfully domesticated uncultured bacteria from environmental samples. The main outcomes of this project will be (1) a new method of cultivating "uncultivable" representatives of human oral microflora, and (2) a collection of novel species potentially important to human health. The newly cultivated species can be readily characterized, i.e. biochemically and genetically (including whole genome sequencing). We will make this collection available to other researchers and will create a free web-based database characterizing this collection. These aims will be achieved in cooperation between three academic labs, one clinical practitioner, and one industrial partner. This multidisciplinary project across five different fields will allow us to achieve the principal goal of the application: gaining access to clinically important members of human oral microflora. Uncultivable bacteria make up more than half of the human oral microflora among which new pathogens or symbionts are likely to be found. We will develop a novel method of isolation and characterization in pure cultures previously uncultivable oral bacteria. This will lead to identifying previously unknown microorganisms that impact human health, and will lay the basis for development of improved therapeutics against oral pathogens.