Cultivating the 'uncultivable' oral microflora

描述（由申请人提供）：口腔中有超过700种细菌，但其中一半以上无法在体外培养，无法用于研究。这项研究的主要目标是获得“不可栽培”的失踪物种。目标1利用一种全新的方法在其自然环境中种植“不可培养物”（Kaeberlein，T.，刘易斯，K.，和Epstein，S. S. 2002.使用模拟的自然环境在纯培养物中分离“不可培养的”微生物（Science 296：1127-1129），结合将生长装置微化至纳米级。纳米室将接种目标微生物的单细胞并原位孵育。纳米室通过限制细胞运动但允许扩散的膜与环境隔开。这将为细胞提供其自然环境的全套生长成分。这绕过了标准培养技术的限制，并且将允许目前未培养的菌株在纯培养物中生长和分离。在目标2中，我们将专注于与口腔疾病有关的物种，并驯化这些物种在标准培养皿中生长。我们以前已经成功地从环境样品中驯化了未培养的细菌。该项目的主要成果将是（1）培养人类口腔微生物菌群“不可培养”代表的新方法，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进行生物化学和遗传学（包括全基因组测序）鉴定。我们将向其他研究人员提供这些收藏，并将创建一个免费的基于网络的数据库来描述这些收藏。这些目标将通过三个学术实验室，一个临床医生和一个工业合作伙伴之间的合作来实现。这个跨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：获得人类口腔微生物群落的临床重要成员。 不可培养的细菌占人类口腔微生物菌群的一半以上，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，分离和表征纯培养以前不可培养的口腔细菌。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.