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: 10327682
• 负责人: Christina A Muzny
• 金额: $ 62.77万
• 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/10327682
• 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; Virulence; Virulent; Woman; adverse outcome; base; 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; 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）是阴道分泌物最常见的原因，与多种不良反应有关。 结果。治疗后复发率>60%，但BV病因仍然未知。BV的特点是 由于阴道乳杆菌的损失以及兼性菌（阴道加德纳菌）和严格厌氧菌的增加。G. 在95-100%的BV病例中存在的迷走神经炎在体外比其他BV相关细菌更具毒性。然而，在这方面， 在具有正常植物群的妇女中也发现了它，并且定殖不足以引起BV的发展。 BV的一个显着特征是阴道上皮细胞上出现多物种生物膜，其中包含 丰富的G. vaginae，较少的Atopobium vaginae和其他未定义的细菌物种。我们假设 G.迷走神经炎启动BV生物膜形成，但偶发BV（iBV）需要将其他关键细菌纳入 改变多微生物聚生体的转录组的生物膜。这与我们的发现一致， 在与女性发生性关系的女性中，两维普雷沃氏菌、G. vagelis和A. iBV前阴道高度依次升高。我们认为这些细菌种类的相似分布 在与男性发生性关系的女性（WSM）中，iBV之前会增加。我们的具体目标是： 目标1.在WSM的纵向研究中调查iBV前阴道微生物群的变化。我们 将从150名具有正常阴道植物群（Nugent评分0-3）的女性中每天两次获得阴道标本，并遵循 iBV（≥4个连续阴道标本的Nugent评分为7-10）持续60天。16 s rRNA基因测序 在iBV前14天，也将在女性中进行靶向V4 [和宽范围16 S rRNA基因qPCR] 保持正常阴道微生物群的年龄相当的女性。 目的2：确定P. bivia对体内多物种BV生物膜的贡献。我们将使用肽 核酸荧光原位杂交（PNA-FISH）探针先前优化G。vagelis和A. 阴道，以分析来自iBV女性的阴道标本的线索细胞中BV生物膜随时间的形成， 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 用于多重使用的探针（Aim 2A）。将对来自女性的样本进行光学显微镜PNA-FISH， iBV和阴性对照（目标2B）。共聚焦激光扫描显微镜FISH将用于确定 在来自iBV女性的线索细胞上的BV生物膜中的3种感兴趣细菌的空间定位（Aim 2C）。 目标3。通过使用RNA测序鉴定与iBV相关的分子标记， 转录组G. bivia和A.阴道我们将探测特定的细菌相互作用， iBV前14天，首先通过RNA测序，使用体外模型在化学成分确定的培养基中模拟 阴道分泌物（mGTS）（目的3A）。文氏图分析将选择最常见的上调基因 在体外试验中共享。潜在的分子标志物与iBV发展的体内相关性将是 通过qPCR评估，使用来自目标1（目标3B）中的iBV女性和阴性对照的阴道样品。