Novel Cultivation Methods for the Domestication of Vaginal Bacteria

阴道细菌驯化的新培养方法

• 批准号: 8291433
• 负责人: DAVID Neal FREDRICKS
• 金额: $ 62.68万
• 依托单位: FRED HUTCHINSON CANCER RESEARCH CENTER
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-08-01 至 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8291433
• 关键词: Address; Aerobic; Affect; Agar; Antibiotics; Appearance; Bacteria; Bacterial Vaginosis; Bathing; Biochemical Pathway; Body Fluids; Body Surface; Body cavities; Cell division; Cells; Chemicals; Coculture Techniques; Communication; Communities; Complex; Culture Media; DNA; Deposition; Devices; Diffusion; Disease; Environment; Epithelial Cells; Extinction (Psychology); Failure; Gardnerella vaginalis; Genital system; Genomics; Goals; Gram' s stain; Growth; Growth Factor; HIV; HIV Infections; Health; Helper-Inducer T-Lymphocyte; Human; In Situ; In Vitro; Incubated; Infection; Laboratories; Lactobacillus; Liquid substance; Mediating; Medical; Membrane; Metabolic; Methods; Microbe; Microbial Genome Sequencing; Microbiology; Molecular; Monitor; Nutrient; Outcome; Participant; Pelvic Inflammatory Disease; Pregnant Women; Premature Birth; Prevotella; Process; Risk; Role; Sampling; Sexually Transmitted Diseases; Signal Transduction; Signaling Molecule; Solid; Source; Sterility; Swab; Tampons; Technology; Time; United States; Vagina; Vaginal Douching; Waste Products; Woman; Women' s Health; adverse outcome; base; body cavity; density; genome sequencing; in vivo; insight; microbiome; migration; miniaturize; neonate; new technology; novel; novel strategies; polycarbonate; prevent; public health relevance; rRNA Genes; repository; routine Bacterial stain; seal; vaginal fluid; volunteer

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.

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）的妇女获得性传播感染（如艾滋病毒）的风险增加，患有BV的孕妇早产的风险增加。我们对BV的理解受到未能培养许多与这种情况相关的细菌的阻碍。PCR方法已经证明，新的和未培养的细菌物种在BV女性中很常见，并且一些未培养的细菌与重要的不良结果相关，例如抗生素治疗失败和上行感染。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，以及介导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人类相关细菌。例如，当细菌物种作为代谢（互养）伙伴一起生长，交换关键营养物质时，单一培养中的生长可能被排除。同样，一个物种可能依赖于第二个物种的信号化合物，刺激细胞分裂，再次排除单一培养。或者，常规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型培养技术和方法来克服这些局限性，这些技术和方法都基于这样的原则，即通过更好地复制天然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小型化扩散室装置在微升规模的室中分离和繁殖细菌。原位分离芯片（iChip）将允许细菌在纯培养物中生长，同时沐浴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，并使用人类阴道本身进行细菌的短期体内繁殖。iChip作为一种中间体，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物中繁殖挑剔的阴道细菌。这种方法使用两个生长室，这两个生长室由可渗透化学物质但不可渗透细菌的膜隔开。将已知培养的细菌或细菌群落接种到下室中，并将未培养的细菌细胞接种到上室中。允许在下部腔室中生长的细菌产生营养物和生长因子以刺激上部腔室中的苛养细菌的增殖。该策略利用细菌物种之间的营养循环和跨物种信号传递来分离难养微生物。在目标3中，我们将使用补充有无菌过滤阴道灌洗液的常规培养基在不同条件下长时间孵育培养难养阴道细菌。该培养基将包含来自阴道环境的缺失的营养物质和信号分子以促进分离。一些新的阴道细菌能够在实验室繁殖，但需要延长的生长时间和非常特定的生长条件。此外，一些细菌可以在实验室中培养，但不容易使用表型方法鉴定。我们将使用16S rRNA基因PCR来帮助鉴定本研究中的所有细菌。 公共卫生相关性：阴道微生物群对妇女和新生儿的健康有重大影响。细菌性阴道病影响美国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物种尚未在实验室中培养。本申请旨在使用几种新的培养方法培养与细菌性阴道病相关的多种挑剔的阴道细菌，目的是获得关于阴道微生物组及其在人类健康中的作用的新见解。