Establishing microbial and biochemical thresholds for development and persistence

摘要 人类阴道中的微生物环境是非常动态的。细菌群落可以转移 戏剧性地从微生物占主导地位，到多样化，多微生物状态，这是一个基本的 细菌性阴道病（BV）。BV是一种与阴道炎相关的临床重要疾病， 以及早产、盆腔炎、术后感染、艾滋病毒的风险增加 和其他性传播感染。不幸的是，人们对促使转变的因素知之甚少 简单和复杂的微生物群落之间，特别是为什么BV通常在以下情况下复发 抗生素治疗本提案的目的是使用多个学科的互补工具， 描述环境因素和种间相互作用对阴道细菌动力学的影响。 我们的重点将是表征BV复发、维持和解决的潜在因素。我们 假设某些物种和营养物质的阈值浓度是BV复发所必需的， 虽然需要不同的条件，特别是生物膜的建立，来维持这种厌氧的， 多微生物状态在目标1中，我们将通过收集阴道拭子来确定细菌动力学的自然史 在女性中，每天3次，持续60天，以用qPCR测量特定细菌种类的丰度。我们将 还收集日常行为日记，并采样pH值和营养生化物质，如血红蛋白和 已知葡萄糖随激素循环而变化。此外，我们还将进行广泛的16S rRNA 基因PCR与焦磷酸测序来描述微生物群落谱多样性的全球变化。我们将 因此能够确定预测阴道突然转变的关键微生物和营养条件 微生物群在目标2中，我们将在选择性条件下测量细菌生长、竞争和生物膜行为。 代谢条件下使用现实的微流体体外培养系统。我们将操纵关键的细菌 代谢物如氨基酸、宿主来源的营养物如葡萄糖和铁、环境条件 如pH值，以及阴道的其他生物物理特征，如剪切力和生物膜形成，以评估 这些变量对微生物生长速率以及多个物种之间微生物竞争的影响 用于补充营养。在目标3中，我们将使用实验室实验（目标2）的结果来填充 数学模型与描述细菌群落动态的竞争性假设。车型将 测试了他们从Aim 1中的人类纵向数据中再现关键动态特征的能力。最优 该模型将为目标1和目标2的观测结果提供机理解释，并将确定条件 对于BV的发展、维持和根除是必要的。更好地理解必要的 微生物突变的条件将为改善生殖健康提供途径。

ABSTRACT The microbial environment in the human vagina is extremely dynamic. Bacterial communities can shift dramatically from lactobacillus predominance, to a diverse, polymicrobial state, which is a fundamental characteristic of bacterial vaginosis (BV). BV is a clinically important condition associated with vaginal discharge as well as heightened risk of preterm birth, pelvic inflammatory disease, post-surgical infections, HIV and other sexually transmitted infections. Unfortunately, little is known about the factors that prompt transitions between simple and complex microbial communities and in particular, why BV commonly recurs following antibiotic treatment. The purpose of this proposal is to use complementary tools from multiple disciplines to describe the influence of environmental factors and interspecies interactions on vaginal bacterial dynamics. Our focus will be on characterizing factors underlying BV recurrence, maintenance and resolution. We hypothesize that threshold concentrations of certain species and nutrients are necessary for recurrent BV, while different conditions, in particular establishment of biofilms, are required for maintenance of this anaerobic, polymicrobial state. In Aim 1 we will define the natural history of bacterial kinetics by collecting vaginal swabs in women 3 times per day for 60 days, to measure abundance of specific bacterial species with qPCR. We will also collect daily behavioral diaries, and sample for pH and nutritive biochemicals such as hemoglobin and glucose which are known to vary with hormonal cycling. In addition, we will perform broad-range 16S rRNA gene PCR with pyrosequencing to describe global changes in microbial community profile diversity. We will therefore be able to identify the key microbial and nutrient conditions that predict abrupt transitions in vagina microbiota. In Aim 2 we will measure bacterial growth, competition, and biofilm behavior under selective metabolic conditions using a realistic microfluidic in vitro cultivation system. We will manipulate key bacterial metabolites such as amino acids, host derived nutrients such as glucose and iron, environmental conditions such as pH, and other biophysical features of the vagina such as shear forces and biofilm formation, to assess the effect of these variable on microbial growth rates, as well as microbial competition among multiple species for scare nutrients. In Aim 3 we will use findings from the laboratory experiments (Aim 2) to populate mathematical models with competing assumptions that describe bacterial community dynamics. Models will be tested for their ability to reproduce key dynamic features from human longitudinal data in Aim 1. The optimal model will generate mechanistic explanations for observations from Aims 1 and 2, and will identify conditions necessary for development, maintenance and eradication of BV. A greater understanding of the necessary conditions for abrupt microbial shifts will offer pathways for improving reproductive health.