Novel Cultivation Methods for the Domestication of Vaginal Bacteria

描述(由申请人提供)：人类阴道的微生物区系可以深刻地影响妇女和新生儿的健康。例如，患有细菌性阴道病(BV)的妇女感染艾滋病毒等性传播疾病的风险增加，BV孕妇早产的风险增加。由于未能培养出许多与这种疾病相关的细菌，我们对BV的了解受到了阻碍。聚合酶链式反应方法表明，新的和未培养的细菌种类在女性BV患者中很常见，一些未培养的细菌与重要的不良后果有关，如抗生素失效和上升感染。微生物基因组测序工作有望为阴道细菌之间的代谢相互作用提供新的见解，这些相互作用有助于维持这些阴道群落，以及调节不良健康结果的关键物种的致病能力。由于各种原因，常规培养方法可能无法繁殖与人类相关的细菌。例如，当细菌物种作为代谢(合养)伙伴一起生长，交换关键的营养物质时，单一培养的生长可能被排除在外。同样，一个物种可能依赖于另一个物种来获得刺激细胞分裂的信号化合物，这也排除了单一培养。或者，传统的实验室介质可能无法复制在体表或体腔中发现的局部环境。我们试图使用几种不同的新的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，即通过更好地复制自然的阴道微环境可以最好地实现阴道细菌的培养。在目标1中，我们将使用一种新型的微型扩散箱设备来在微升规模的箱中分离和繁殖细菌。原位分离芯片(IChip)将允许细菌在纯培养中生长，同时沐浴在来自自然环境的液体中。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，而人体阴道本身则进行短期的体内细菌繁殖。IChip是一种中间体，用于使细菌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。这种方法使用两个生长室，由一层对化学物质可渗透但细菌不能渗透的膜隔开。已知的培养细菌或细菌群落被接种到下室中，而未培养的细菌细胞被接种到上室中。在下室中生长的细菌被允许产生营养物质和生长因子，以刺激上室中挑剔的细菌的繁殖。这一策略利用营养循环和细菌物种之间的跨物种信号传递来分离挑剔的微生物。在目标3中，我们将使用常规培养基加无菌过滤阴道灌洗液，在不同条件下培养挑剔的阴道细菌，并延长培养时间。这种培养基会包含阴道环境中缺失的营养物质和信号分子，以便于隔离。一些新的阴道细菌能够在实验室中繁殖，但需要延长生长时间和非常特殊的生长条件。此外，一些细菌可以在实验室培养，但不容易使用表型方法进行鉴定。本研究采用16S rRNA基因聚合酶链式反应(16S RRNA)对所有细菌进行鉴定。 公共卫生相关性：阴道微生物区系对妇女和新生儿的健康有重大影响。细菌性阴道病在美国影响着29%的妇女，并与性传播疾病的风险增加有关，包括艾滋病毒、早产、盆腔炎和其他几种不良后果。细菌性阴道病的微生物学知之甚少，在这种情况下发现的许多细菌种类还没有在实验室培养出来。这项应用寻求使用几种新的培养方法培养与细菌性阴道病相关的大量挑剔的阴道细菌，目的是获得对阴道微生物及其在人类健康中的作用的新见解。

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.