The Role of Uncultivated Bacteria in Bacterial Vaginosis

描述（由申请人提供）：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，并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，但阴道植物群的变化是值得注意的，患者可能对抗生素治疗有反应。没有单一的培养细菌与BV特异性相关。复杂微生物群落中的细菌可以通过表征核糖体DNA序列而无需培养来鉴定。这种方法提供了检测挑剔或耐培养微生物的优势。该项目的短期目标是使用培养独立的分子方法来表征BV女性和健康对照的阴道植物群。长期的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。为了实现这些目标，将追求以下目标：目标1：确定与BV相关的细菌群落。将使用宽范围PCR扩增阴道液中存在的细菌16S rRNA基因。扩增的16S rDNA的序列分析将用于鉴定BV受试者和健康对照中发现的细菌菌种。目的2：利用细菌特异性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检测比宽范围PCR更敏感，用于检测复杂群落中的罕见细菌物种。这些方法将应用于其他BV受试者和健康对照的阴道液样本。FISH与荧光显微镜将用于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：确定是否存在BV相关细菌的阴道外小生境。胃肠道是许多厌氧菌的宿主，可能是BV相关细菌的储存库。将从有和无BV的女性中获得阴道外粘膜表面拭子。将使用细菌特异性PCR检测这些拭子上的细菌。我们假设BV相关细菌将是人类胃肠道的常见定植者。从该项目中获得的知识将有助于识别BV受试者中发现的细菌群落，将为通过新的PCR和FISH检测推进BV诊断提供机会，并可能有助于通过识别阴道外感染储库来预防BV。

DESCRIPTION (provided by applicant): 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 16S rRNA genes present in vaginal fluid. Sequence analysis of the amplified 16S rDNA will be used to identify the bacterial species found in subjects with BV and healthy controls. Aim 2: Use bacterium specific PCR and fluorescence in situ hybridization (FISH) assays to detect and quantify each vaginal bacterium. Bacterium-specific PCR 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. Aim 3: Determine if there is an extra-vaginal niche for BV associated bacteria. The gastrointestinal tract hosts numerous anaerobic bacteria and may be a reservoir for the bacteria linked to BV. Swabs of extra-vaginal mucosal surfaces will be obtained from women with and without BV. Bacteria on these swabs will be detected using bacterium-specific PCRs. We hypothesize that BV-associated bacteria will be common colonizers of the human 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.