Microbial Interactions between Gardnerella, Prevotella, and Atopobium Prior to Incident Bacterial Vaginosis

细菌性阴道病发生前加德纳菌、普雷沃菌和 Atopobium 之间的微生物相互作用

• 批准号: 10559570
• 负责人: Christina A Muzny
• 金额: $ 61.73万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-02-01 至 2025-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10559570
• 关键词: 16S ribosomal RNA sequencing; Address; Age; Anaerobic Bacteria; Appearance; Atopobium vaginae; Bacteria; Bacterial Vaginosis; Cells; Chemicals; Development; Diagnosis; Epithelial Cells; Etiology; Exhibits; Fluorescent in Situ Hybridization; Gardnerella; Gardnerella vaginalis; Genes; HIV; Hela Cells; In Vitro; Lactobacillus; Laser Scanning Confocal Microscopy; Longitudinal Studies; Menstrual cycle; Microbial Biofilms; Organism; Pathogenesis; Pathway Analysis; Pelvic Inflammatory Disease; Peptide Nucleic Acids; Premature Birth; Prevention; Prevotella; Public Health; Recurrence; Research; Risk; Sampling; Sexual Transmission; Sexually Transmitted Diseases; Specimen; Time; Vagina; Vaginal Discharge; Virulent; Woman; adverse outcome; design; epidemiologic data; improved; in vitro Assay; in vitro Model; in vivo; interest; light microscopy; men; microbiome analysis; microorganism interaction; molecular marker; normal microbiota; pathogen; polymicrobial biofilm; rRNA Genes; sex; transcriptome; transcriptome sequencing; vaginal lactobacilli; vaginal microbiota; virulence gene; women who have sex with women

ABSTRACT: Bacterial vaginosis (BV), the most common cause of vaginal discharge, is associated with multiple adverse outcomes. The rate of recurrence after therapy is >60% yet BV etiology remains unknown. BV is characterized by loss of vaginal lactobacilli and increases in facultative (Gardnerella vaginalis) and strict anaerobes. G. vaginalis, present in 95-100% of BV cases, is more virulent than other BV-associated bacteria in vitro. However, it is also found in women with normal flora and colonization is not sufficient for BV development. A notable feature of BV is the appearance of a multi-species biofilm on vaginal epithelial cells containing abundant G. vaginalis, fewer Atopobium vaginae, and other undefined bacterial species. We hypothesize that G. vaginalis initiates BV biofilm formation, but incident BV (iBV) requires incorporation of other key bacteria into the biofilm that alter the transcriptome of the polymicrobial consortium. This is consistent with our finding that, among women who have sex with women, the mean relative abundance of Prevotella bivia, G. vaginalis, and A. vaginae became sequentially higher prior to iBV. We propose that a similar distribution of these bacterial species will increase prior to iBV in women who have sex with men (WSM). Our specific aims are: Aim 1. Investigate changes in the vaginal microbiota preceding iBV in a longitudinal study of WSM. We will obtain twice daily vaginal specimens from 150 women with normal vaginal flora (Nugent score 0-3) and follow them for iBV (Nugent score 7-10 on ≥4 consecutive vaginal specimens) for 60 days. 16S rRNA gene sequencing targeting V4 [and broad range 16S rRNA gene qPCR] will be done in women for the 14 days prior to iBV as well as age-comparable women maintaining normal vaginal microbiota. Aim 2: Determine the contribution of P. bivia to the multi-species BV biofilm in vivo. We will use peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) probes previously optimized for G. vaginalis and A. vaginae to analyze BV biofilm formation over time in clue cells from vaginal specimens from women with iBV in Aim 1, focusing on the 14 days prior to iBV. We will design, validate, and optimize a new P. bivia PNA-FISH probe for multiplex use (Aim 2A). Light microscopy PNA-FISH will be performed on specimens from women with iBV and negative controls (Aim 2B). Confocal laser scanning microscopy FISH will be used to determine the interspatial localization of the 3 bacteria of interest in the BV biofilm on clue cells from women with iBV (Aim 2C). Aim 3. Identify molecular markers associated with iBV by using RNA sequencing to analyze the transcriptome of G. vaginalis, P. bivia, and A. vaginae. We will probe specific bacterial interactions during the 14 days prior to iBV, first by RNA sequencing using in vitro models in a chemically defined medium simulating vaginal secretions (mGTS) (Aim 3A). Venn-diagram analysis will select the most commonly up-regulated genes shared in the in vitro assays. The in vivo relevance of potential molecular markers for development of iBV will be assessed by qPCR, using vaginal samples from women with iBV and negative controls in Aim 1 (Aim 3B).

摘要： 细菌性阴道病(BV)是导致阴道分泌物的最常见原因，与多种不良反应有关。 结果。治疗后的复发率为60%，但BV的病因尚不清楚。BV的特点是 由于阴道乳杆菌的丧失和兼性(阴道加德纳氏菌)和严格的厌氧菌的增加。G. 阴道杆菌存在于95-100%的BV病例中，在体外比其他BV相关细菌的毒力更强。然而， 在具有正常菌群的妇女中也发现了这种情况，而定居不足以促进BV的发展。 BV的一个显著特征是在阴道上皮细胞上出现多物种生物film，该细胞含有 丰富的阴道革兰氏菌，较少的阴道阿托波菌，以及其他未知的细菌种类。我们假设 阴道毛滴虫启动BV生物被膜的形成，但偶发的BV(IBV)需要将其他关键细菌整合到 改变多菌体转录组的生物膜。这与我们的发现是一致的， 在与女性发生性关系的女性中，比普氏原虫、阴道革兰氏原虫和A。 在IBV之前，阴道依次升高。我们认为，这些细菌物种的相似分布 在与男性发生性行为的女性(WSM)感染IBV之前将会增加。我们的具体目标是： 目的1.在WSM的纵向研究中，调查IBV前阴道微生物区系的变化。我们 将从150名阴道菌群正常(纽金特评分0-3)的女性身上每天采集两次阴道样本，并跟踪 用≥连续4次取阴道标本检测鸡传染性支气管炎病毒(纽金特评分7~10分)，为期60天。16S rRNA基因测序 针对V4[和广泛范围的16S rRNA基因qPCR]的女性也将在IBV前14天进行 与年龄相当的女性保持正常的阴道微生物区系。 目的2：确定P.bivia对体内多菌种BV生物被膜的贡献。我们将使用多肽 核酸荧光原位杂交(PNA-FISH)探针对阴道毛滴虫和毛滴虫进行了优化。 阴道分析BV生物膜的形成随时间的变化线索细胞从阴道标本从妇女IBV年 目标1，重点关注IBV前14天。我们将设计、验证和优化一种新的P.bivia PNA-FISH 用于多用途的探测器(目标2A)。光镜PNA-FISH将对患有 IBV和阴性对照(目标2B)。将使用激光共聚焦扫描显微镜FISH来确定 BV生物膜中感兴趣的3种细菌在IBV妇女线索细胞上的空间定位(目标2C)。 目的3.利用RNA测序技术寻找与IBV相关的分子标记 阴道毛滴虫、阴道毛滴虫和阴道毛滴虫的转录组。我们将探索特定的细菌相互作用在 IBV前14天，首先在化学定义的模拟介质中使用体外模型进行RNA测序 阴道分泌物(MGTS)(目标3A)。维恩图分析将选择最常见的上调基因 在体外试验中也是一样的。IBV发展的潜在分子标志物的体内相关性将是 在目标1(目标3B)中，使用来自IBV患者和阴性对照的阴道样本，通过定量聚合酶链式反应进行评估。