The Role of Uncultivated Bacteria in Bacterial Vaginosis

细菌性阴道病（BV）是一种影响数百万妇女的疾病，产生恶臭的阴道分泌物， 与几种严重的健康状况有关，包括早产和艾滋病毒感染。BV的原因尚不清楚， 尽管注意到阴道植物群的变化，并且患者可能对抗生素治疗有反应。没有单一的栽培 细菌与BV特异性相关。复杂微生物群落中的细菌可以在没有 通过表征核糖体DNA序列进行培养。这种方法提供了检测挑剔或 耐培养微生物。本项目的短期目标是利用非培养依赖性分子生物学技术， 方法描述BV患者和健康对照组的阴道植物群。长期目标是 了解阴道生态位的细菌生态，并确定未培养的细菌在BV中的作用。 假设：在BV受试者中发现了新的细菌群落，包括许多未培养的菌种。到 为了实现这些目标，将追求以下目标：目标1：确定与 BV.将使用宽范围PCR扩增阴道液中存在的细菌16SrRNA基因。序列分析 扩增的16SrDNA将用于鉴定BV受试者和健康对照中发现的细菌种类。 目的2：细菌特异性PCR和荧光原位杂交（FISH）检测和定量 阴道细菌细菌特异性PCR和FISH检测比广谱PCR更敏感， 复杂群落中罕见的细菌物种。这些方法将应用于阴道液样本， 其他BV受试者和健康对照。将使用FISH与荧光显微镜配对进行计数 阴道液涂片中的细菌，从而产生细菌代表性的独立测量，以补充PCR 方法.目的3：确定BV相关细菌是否存在阴道外小生境。胃肠道 宿主大量厌氧菌，可能是BV相关细菌的储存库。阴道外拭子 粘膜表面将从患有和不患有BV的女性获得。这些拭子上的细菌将使用 细菌特异性PCR。我们假设BV相关细菌将是人类的常见定植者， 胃肠道从这个项目中获得的知识将有助于确定在受试者中发现的细菌群落 与BV，将产生机会，以推进BV的诊断，通过新的PCR和FISH检测，并可能有助于 通过识别阴道外感染库来预防BV。

Bacterial vaginosis (BV) is a condition that affects millions of women, produces malodorous vaginal discharge, and is linked to several serious health conditions including preterm labor and HIV infection. The cause of BV is unclear, though changes in vaginal flora are noted and patients may respond to antibiotic treatment. No single cultivated bacterium is specifically associated with BV. Bacteria in complex microbial communities can be identified without cultivation by characterizing ribosomal DNA sequences. This approach offers the advantage of detecting fastidious or cultivation-resistant microbes. The short-term objective of this project is to use cultivation-independent molecular methods to characterize the vaginal flora of women with BV and healthy controls. The long-term objectives are to understand the bacterial ecology of the vaginal niche and determine the role uncultivated bacteria play in BV. Hypothesis: novel communities of bacteria, including many uncultivated species, are found in subjects with BV. To meet these objectives, the following aims will be pursued: Aim 1:Identify the community of bacteria associated with BV. Broad range PCR will be used to amplify bacterial 16SrRNA genes present in vaginal fluid. Sequence analysis of the amplified 16SrDNA will be used to identify the bacterial species found in subjects with BV and healthycontrols. Aim 2: Usebacterium specific PCR and fluorescence in situ hybridization (FISH) assays to detect and quantify each vaginal bacterium. Bacterium-specificPCR and FISH assays are more sensitive than broad range PCR for detecting infrequent bacterial species in complex communities. These methods will be applied to vaginal fluid samples from additional subjects with BV and healthy controls. FISH paired with fluorescence microscopy will be used to count bacteria in vaginal fluid smears, thus producing an independent measure of bacterial representation to complement PCR methods. Aim3:Determine if there is an extra-vaginal niche for BVassociated bacteria. The gastrointestinal tract hosts numerous anaerobic bacteria and may be a reservoir for the bacteria linked to BV. Swabs ofextra-vaginal mucosal surfaces will be obtained from women with and without BV. Bacteria on these swabs will be detectedusing bacterium-specific PCRs. We hypothesize that BV-associated bacteria will be common colonizers of thehuman gastrointestinal tract. Knowledge gained from this project will help identify the bacterial communities found in subjects with BV, will generate opportunities to advance the diagnosis of BV through new PCR and FISH assays, and may help in the prevention of BV by identifying extra-vaginal reservoirs of infection.