Novel Cultivation Methods for the Domestication of Vaginal Bacteria

描述（由申请人提供）：人类阴道的微生物群可以深刻影响妇女和新生儿的健康。例如，患有细菌性阴道病（BV）的妇女获得艾滋病毒等性传播感染的风险增加，患有细菌性阴道病的孕妇早产的风险增加。我们对细菌性阴道炎的了解由于未能培养出许多与此病相关的细菌而受到阻碍。PCR方法表明，新的和未培养的细菌种类在细菌性阴道炎妇女中很常见，一些未培养的细菌与抗生素失效和上升感染等重要不良后果有关。微生物基因组测序工作有望提供有关阴道细菌之间代谢相互作用的新见解，这些细菌有助于维持这些阴道群落和介导不良健康结果的关键物种的致病能力。由于各种原因，传统的培养方法可能无法繁殖与人类相关的细菌。例如，当细菌种类作为代谢（合养）伙伴一起生长，交换关键营养物质时，在单一栽培中生长可能会被阻止。同样，一个物种可能依赖另一个物种来获得刺激细胞分裂的信号化合物，这再次排除了单一培养。另外，传统的实验室培养基可能无法复制体表或体腔中的局部环境。我们试图利用几种不同的新型培养技术和方法来克服这些限制，这些技术和方法都基于这样的原则，即阴道细菌的培养可以通过更好地复制自然阴道微环境来实现。在Aim 1中，我们将使用一种新型的小型化扩散室装置在微升规模的室中分离和繁殖细菌。这种原位分离芯片（iChip）可以让细菌在纯培养物中生长，同时浸泡在自然环境中的液体中。在这种情况下，我们将使用阴道灌洗液进行细菌的体外繁殖，并使用人体阴道本身进行细菌的短期体内繁殖。芯片作为一种媒介，使细菌适应在实验室培养基上独立生长，这一过程我们称之为驯化。在目标2中，我们将使用共培养方法在纯培养中繁殖挑剔的阴道细菌。这种方法使用两个由膜隔开的生长室，膜可以通过化学物质，但不能通过细菌。将已知培养的细菌或细菌群落接种于下室，将未培养的细菌细胞接种于上室。让生长在下腔的细菌产生营养物质和生长因子，刺激上腔挑剔细菌的增殖。这种策略利用了养分循环和细菌物种之间的跨物种信号，以分离挑剔的微生物。在Aim 3中，我们将使用常规培养基补充无菌过滤阴道灌洗液，在不同条件下培养挑剔的阴道细菌，并延长孵育时间。这种介质将含有阴道环境中缺失的营养物质和信号分子，以促进隔离。一些新的阴道细菌能够在实验室繁殖，但需要延长生长时间和非常特殊的生长条件。此外，有些细菌可以在实验室培养，但不容易用表型方法鉴定。我们将使用16S rRNA基因PCR技术来方便本研究中所有细菌的鉴定。

DESCRIPTION (provided by applicant): The microbiota of the human vagina can profoundly affect the health of women and neonates. For instance, women with the condition bacterial vaginosis (BV) have increased risks of acquiring sexually transmitted infections such as HIV, and pregnant women with BV have increased risk of preterm birth. Our understanding of BV is hampered by the failure to cultivate many of the bacteria associated with this condition. PCR methods have demonstrated that novel and uncultivated bacterial species are common in women with BV and some uncultivated bacteria are associated with important adverse outcomes such as antibiotic failure and ascending infection. Microbial genome sequencing efforts hold the promise of providing new insights regarding the metabolic interactions among vaginal bacteria that help sustain these vaginal communities and the pathogenic capabilities of key species that mediate poor health outcomes. Conventional cultivation methods may fail to propagate human-associated bacteria for a variety of reasons. For example, growth in monoculture may be precluded when bacterial species grow together as metabolic (syntrophic) partners, exchanging critical nutrients. Likewise, one species may depend on a second species for a signaling compound that stimulates cell division, again precluding monoculture. Alternatively, conventional laboratory medium may fail to replicate the local environment found in a body surface or cavity. We seek to overcome these limitations using several different novel cultivation technologies and approaches, which are all based on the principle that cultivation of vaginal bacteria can best be achieved by better reproducing the natural vaginal microenvironment. In Aim 1 we will use a novel miniaturized diffusion chamber device to isolate and propagate bacteria in microliter scale chambers. The in situ isolation chip (iChip) will allow bacteria to grow in pure culture while bathed in fluid from the natural environment. In this case, we will use vaginal lavage fluid for in vitro propagation of bacteria, and the human vagina itself for short term in vivo propagation of bacteria. The iChip serves as an intermediate for adapting bacteria for independent growth on laboratory media, a process that we call domestication. In Aim 2, we will use co-cultivation methods to propagate fastidious vaginal bacteria in pure culture. This approach uses two growth chambers that are separated by a membrane permeable to chemicals but not bacteria. A known cultivated bacterium or bacterial community is inoculated into the lower chamber, and an uncultivated bacterial cell is inoculated in the upper chamber. Bacteria growing in the lower chamber are allowed to produce nutrients and growth factors to stimulate the proliferation of fastidious bacteria in the upper chamber. This strategy takes advantage of nutrient cycling and cross species signaling between bacterial species to allow isolation of fastidious microbes. In Aim 3, we will use conventional media supplemented with sterile filtered vaginal lavage fluid to cultivate fastidious vaginal bacteria under diverse conditions with prolonged incubation. This media will contain missing nutrients and signaling molecules from the vaginal environment to facilitate isolation. Some novel vaginal bacteria are capable of laboratory propagation but require extended growth times and very specific growth conditions. Furthermore, some bacteria can be cultivated in the lab but are not easily identified using phenotypic methods. We will use 16S rRNA gene PCR to facilitate identification of all bacteria in this study. PUBLIC HEALTH RELEVANCE: The vaginal microbiota has a major impact on the health of women and neonates. Bacterial vaginosis affects 29% of women in the United States and is associated with increased risk of sexually transmitted diseases, including HIV, preterm birth, pelvic inflammatory disease, and several other adverse outcomes. The microbiology of bacterial vaginosis is poorly understood and many bacterial species found in this condition have not been cultivated in the laboratory. This application seeks to cultivate numerous fastidious vaginal bacteria associated with bacterial vaginosis using several novel cultivation approaches with the goal of gaining new insights about the vaginal microbiome and its role in human health.