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

基本信息

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
项目类别：
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

项目摘要

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).

抽象的：细菌性阴道病（BV）是阴道分泌物最常见的原因，与多种不良症状有关治疗后复发率>60%，但 BV 病因仍不清楚。阴道乳酸菌的减少和兼性（阴道加德纳菌）和严格厌氧菌的增加。阴道念珠菌存在于 95-100% 的 BV 病例中，在体外比其他 BV 相关细菌的毒性更强。它也存在于具有正常菌群的女性中，并且定植不足以促进 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，其中含有丰富的 G. vaginalis，较少的 Atopobium vaginae 和其他未定义的细菌种类。 G. vaginalis 启动 BV 生物膜形成，但意外 BV (iBV) 需要将其他关键细菌掺入改变多微生物群落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，普雷沃氏菌、阴道加德氏菌和阿氏杆菌的平均相对丰度。我们认为这些细菌种类的分布在 iBV 之前依次升高。在与男性发生性关系的女性 (WSM) 中，iBV 会增加。我们的具体目标是：目标 1. 在 WSM We 的纵向研究中调查 iBV 之前阴道微生物群的变化。将从 150 名具有正常阴道菌群（Nugent 评分 0-3）的女性那里获取每天两次的阴道标本，并遵循对他们进行 60 天的 iBV（≥4 个连续阴道标本的 Nugent 评分 7-10）。针对 V4 [和广泛的 16S rRNA 基因 qPCR] 也将在 iBV 前 14 天在女性中进行与年龄相当的女性保持正常的阴道微生物群一样。目标 2：确定 P. bivia 对体内多物种 BV 生物膜的贡献我们将使用肽。之前针对 G. vaginalis 和 A. vaginalis 进行了优化的核酸荧光原位杂交 (PNA-FISH) 探针。阴道，分析 iBV 女性阴道标本线索细胞中 BV 生物膜随时间的形成情况目标 1，重点关注 iBV 之前的 14 天，我们将设计、验证和优化新的 P. bivia PNA-FISH。多重使用的探针（目标 2A）将对来自女性的标本进行光学显微镜 PNA-FISH。 iBV 和阴性对照 (Aim 2B) 将使用共焦激光扫描显微镜 FISH 来确定。 BV 生物膜中 3 种感兴趣的细菌在 iBV 女性线索细胞上的空间定位（目标 2C）。目标 3. 通过使用 RNA 测序来分析与 iBV 相关的分子标记我们将探究 G. vaginalis、P. bivia 和 A. vaginae 的转录组。 iBV 前 14 天，首先使用体外模型在化学成分确定的培养基中进行 RNA 测序，模拟阴道分泌物 (mGTS)（目标 3A）维恩图分析将选择最常见上调的基因。 iBV 发展的潜在分子标记的体内相关性将在体外测定中共享。使用目标 1（目标 3B）中的 iBV 女性阴道样本和阴性对照通过 qPCR 进行评估。