Cultivation and domestication of previously uncultivated species from human oral

描述(申请人提供)：1898年，奥地利微生物学家海因里希·温特伯格进行了一项奇怪的观察：他样本中的微生物细胞数量与营养培养基上形成的菌落数量不匹配(温特伯格1898)。在十年内，这种不匹配被量化，并被证明是惊人的大：可培养细胞的百分比被证明不到1%。这标志着朝着今天所知的大平板计数异常(Staley和Konopka，1985)这一重要发现迈出的最早的一步，可以说是最古老的未解决的微生物现象。这项建议从临床表现上解决了这一现象。来自人类微生物组的微生物，包括口腔，培养能力很差：超过50%的口腔微生物区系仍未从培养物集合中找到。这是对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，已与各种系统性疾病有关。在这里，我们提出了两种方法来驯化以前未培育的物种从人类口腔。这两种方法都大大偏离了标准的栽培技术，并解决了它们的一些局限性。我们在对来自水环境和土壤的未培养微生物的环境研究中成功地测试了这些方法，并在这里对它们进行了调整，以满足临床需求。在目标1中，我们采用了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，防止了杂草物种的过度生长，而长期(几周)的培养为生长较慢的生物体提供了适当的条件。总而言之，这给了一个生长(并被检测到)到任何细胞的机会，无论分裂多么缓慢或罕见。为了解释额外的生长偏好，我们对该方法进行了两次修改，分别基于液体和固体介质。令人鼓舞的是，我们最近将这种方法应用于环境微生物，分离出了几个非常新的物种。在目标2中，我们利用了这样的观察，即在自然界中，微生物物种不是孤立生长的，而是经常作为微生物联合体的相互依赖的组成部分。微生物之间的生态相互作用有许多方面，从碳源的共代谢，到代谢产物的交换，再到物种间的信号传递。这种相互作用，以及由此产生的生长过程中的相互依赖，可能会使一些物种在失去自然协同效应的情况下无法分裂。我们开发了一种微生物共培养的方法，并成功地将其应用于水生和土壤新物种的培养。我们还设计了一种简单的技术来将微生物联合体分离成它们的组成物种。这使得研究伙伴物种提供的益处成为可能，目的是通过用联合体内交换的化合物(S)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是本研究的天然产物化学成分。我们将询问来自Aim 2的微生物联盟，因为它们对临床研究很重要，并研究微生物共培养中生长相互依赖的化学基础。利用生物测定引导的分级，再加上最先进的微尺度LC-MS和核磁共振分析，我们将确定财团内负责诱导/刺激生长的因素的化学结构。然后，我们将用这些因素来补充标准媒体，使它们能够独立于目标物种的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计划。我们成功地利用环境协会进行了一项概念研究，并将这些经验用于口腔微生物区系研究。我们注意到，这里使用和开发的所有方法本质上都是一般性的，并将很容易适用于对人类微生物组其他部分的研究。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控制和预防的进展。 与公共健康相关：微生物学中最重要和最耐人寻味的观察之一是，只有少数物种在实验室中生长。在很大程度上，人文微生物组，包括口腔微生物区系，仍然没有培养，无论是基础研究还是临床研究都无法获得。在这个项目中，我们将开发两种新的和通用的方法，基于单细胞培养和协同物种的共培养，这将导致临床上重要的新物种的分离和驯化。

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.