Cultivation and domestication of previously uncultivated species from human oral

从人类口腔中培育和驯化以前未培育的物种

• 批准号: 7933992
• 负责人: Slava Simon Epstein
• 金额: $ 45.06万
• 依托单位: NORTHEASTERN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-17 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7933992
• 关键词: Accounting; Address; Arts; Biological Assay; Biomass; Carbon; Cell Count; Cells; Chemical Structure; Chemicals; Chemistry; Clinical; Clinical Microbiology; Clinical Research; Coculture Techniques; Codependence; Collection; Communities; Coupled; Data; Databases; Dependence; Deposition; Devices; Disease; Environment; Epithelial Cells; Exhibits; Fractionation; Future; Glass; Goals; Growth; Growth Factor; Guanine Nucleotide Exchange Factors; Health; Human; Human Microbiome; Human body; In Vitro; Incubated; Individual; Investigation; Lead; Liquid substance; Masks; Metabolism; Methodology; Methods; Microbe; Microbiologic Phenomena; Microbiology; Modification; Mouth Diseases; Natural Products Chemistry; Nature; Nutrient; Oral; Oral cavity; Organism; Prevention; Recovery; Research; Research Personnel; Sampling; Series; Signal Transduction; Slide; Soil; Solid; Source; System; Systemic disease; Techniques; Technology; Testing; Time; Tooth structure; base; design; disorder control; experience; interest; member; microbial; microbiome; microorganism; microorganism interaction; novel; novel strategies; oral bacteria; oral microbiome; preference; prevent; programs; public health relevance; research study

DESCRIPTION (provided by applicant): In 1898, an Austrian microbiologist Heinrich Winterberg made a curious observation: the number of microbial cells in his samples did not match the number of colonies formed on nutrient media (Winterberg 1898). Within a decade, this mismatch was quantified, and turned out to be surprisingly large: the percent of cultivable cells proved to be less than 1%. This signified one of the earliest steps towards an important discovery known today as the Great Plate Count Anomaly (Staley and Konopka 1985), - arguably the oldest unresolved microbiological phenomenon. This proposal addresses this phenomenon in its clinical manifestation. Microorganisms from the human microbiome, including the oral cavity, are poorly cultivable: over 50% of oral microflora are still missing from culture collections. This is a significant impediment to the study of human health because oral bacteria, including uncultivated species, have been implicated in a variety of systemic diseases. Here we propose two approaches to domesticate previously uncultivated species from the human oral cavity. Both approaches represent a substantial departure from standard cultivation techniques, and address some of their limitations. We successfully tested these approaches in our environmental research on uncultivated microorganisms from aquatic environment and soils, and adapt them here to address clinical needs. In Aim 1, we employ a long-term, single cell cultivation method. Single cell cultivation eliminates competition from other strains and prevents overgrowing by weed species, whereas long-term (weeks) incubation provides appropriate conditions for slower growing organisms. Collectively, this gives a chance to grow (and be detected) to any cell, no matter how slowly dividing or rare. To account for additional growth preferences, we developed two modifications of the method, based on liquid and solid media. It is encouraging that our recent application of this method to environmental microorganisms led to isolation of several remarkably novel species. In Aim 2, we utilize the observation that, in nature, microbial species do not grow in isolation but often as codependent components of microbial consortia. The ecological interactions between microbial partners have many aspects, from co-metabolism of a carbon source, to exchange of metabolites, to interspecies signaling. Such interactions, and the resulting co-dependence in growth, may make some species unable to divide if deprived of their natural synergies. We developed a method for co-cultivating microorganisms, and successfully applied it to cultivating novel aquatic and soil species. We also designed a simple technique to separate the microbial consortia into their component species. This enables investigation of the benefits provided by the partner species, with the goal of mimicking the synergistic requirements by supplementing the medium with the compound(s) exchanged within consortia. This possibility is explored in Aim 3. Aim 3 is the natural product chemistry component of the study. We will interrogate microbial consortia from Aim 2, chosen for their importance to clinical research, and investigate the chemical underpinning of growth codependence in microbial co-cultures. Using bioassay-guided fractionations, coupled with state-of-the-art microscale LC-MS and NMR analyses, we will determine the chemical structure of factors responsible for growth induction/stimulation within consortia. We will then supplement standard media with these factors, making them capable of supporting growth of the target species independently of their synergistic partners. This will domesticate these species and make them available to clinical research programs. We successfully conducted a concept study using environmental consortia, and utilize these experiences here for the purposes of oral microflora research. We note that all methodologies used and developed here are general in nature, and will be readily applicable to studies of other compartments of the human microbiome. We will share these technologies with the community, and make strains obtained here available to others to further progress in oral disease control and prevention. PUBLIC HEALTH RELEVANCE: One of the most important and intriguing observation in microbiology is that only few species grow in the lab. For the most part, the humane microbiome, including oral microflora, remains uncultivated and inaccessible for either basic or clinical research. In this project, we will develop two novel, and general, approaches based on single cell cultivation as well as co-cultivation of synergistic species, which will lead to isolation and domestication of novel species of clinical importance.

描述（由申请人提供）：1898年，一位奥地利微生物学家Heinrich Winterberg做了一个奇怪的观察：他的样品中微生物细胞的数量与营养培养基上形成的菌落数量不匹配（Winterberg 1898）。十年之内，这种不匹配被量化，结果证明其大得惊人：可培养细胞的百分比被证明小于 1%。这标志着今天被称为“大板块计数异常”（Staley 和 Konopka 1985）的重要发现的最早步骤之一，可以说是最古老的未解决的微生物现象。该提案解决了这一现象的临床表现。来自人类微生物群（包括口腔）的微生物很难培养：超过 50% 的口腔微生物群在培养物保藏中仍然缺失。这是人类健康研究的一个重大障碍，因为口腔细菌，包括未培养的物种，与多种全身性疾病有关。在这里，我们提出了两种从人类口腔驯化以前未培养的物种的方法。这两种方法都与标准栽培技术有很大的不同，并解决了它们的一些局限性。我们在水生环境和土壤中的未培养微生物的环境研究中成功测试了这些方法，并在此进行调整以满足临床需求。在目标 1 中，我们采用长期的单细胞培养方法。单细胞培养消除了来自其他菌株的竞争，并防止杂草物种过度生长，而长期（数周）培养则为生长缓慢的生物体提供了适当的条件。总的来说，这为任何细胞提供了生长（并被检测到）的机会，无论分裂多么缓慢或罕见。为了考虑到额外的生长偏好，我们基于液体和固体培养基对该方法进行了两种修改。令人鼓舞的是，我们最近将这种方法应用于环境微生物，分离出了几种非常新的物种。在目标 2 中，我们利用了这样的观察结果：在自然界中，微生物物种并不是孤立生长的，而是通常作为微生物群落的相互依赖的组成部分。微生物伙伴之间的生态相互作用有很多方面，从碳源的共同代谢，到代谢物的交换，再到种间信号传导。这种相互作用以及由此产生的生长相互依赖，如果剥夺了它们的自然协同作用，可能会使一些物种无法分裂。我们开发了一种微生物共培养方法，并成功应用于培育新的水生和土壤物种。我们还设计了一种简单的技术将微生物群落分成其组成物种。这使得能够研究伙伴物种提供的益处，目的是通过用联盟内交换的化合物补充介质来模拟协同需求。目标 3 探讨了这种可能性。目标 3 是该研究的天然产物化学组成部分。我们将询问目标 2 中的微生物群落（因其对临床研究的重要性而被选择），并研究微生物共培养物中生长相互依赖性的化学基础。使用生物测定引导的分馏，再加上最先进的微型 LC-MS 和 NMR 分析，我们将确定负责联盟内生长诱导/刺激的因子的化学结构。然后，我们将用这些因子补充标准培养基，使它们能够独立于其协同伙伴而支持目标物种的生长。这将驯化这些物种并使它们可用于临床研究项目。我们利用环境联盟成功地进行了一项概念研究，并将这些经验用于口腔微生物群研究。我们注意到，这里使用和开发的所有方法本质上都是通用的，并且很容易适用于人类微生物组其他区室的研究。我们将与社区分享这些技术，并将在这里获得的菌株提供给其他人，以进一步推动口腔疾病的控制和预防。 公共卫生相关性：微生物学中最重要和最有趣的观察之一是实验室中生长的物种很少。在大多数情况下，包括口腔微生物群在内的人道微生物群仍未被培养，无法用于基础或临床研究。在这个项目中，我们将开发两种基于单细胞培养以及协同物种共培养的新颖且通用的方法，这将导致具有临床重要性的新物种的分离和驯化。