Adaptation of vancomycin-resistant enterococci during bloodstream infection

摘要 该项目长期目标是了解万古霉素耐药肠球菌(VRE)如何适应 在血流感染期间(BSI)，以更好地耐受抗生素和宿主免疫防御。肠球菌有 经过数亿年的进化，在动物的胃肠道(GI)上定居，它们很好 适应了那里的生活。然而，引起BSI的VRE面临着本质上不同的选择压力，例如 高浓度的抗生素、营养限制和宿主免疫防御。在过去的五年里在我们的中心 多年来，VRE-BSI患者的30天死亡率为36%，高于BSI，原因是所有其他因素 ESKAPE病原体。此外，VRE-BSI通常难以治疗，近三分之一的VRE-BSI患者- BSI经历菌血症延长(≥5天)，或在一年内反复感染。在这里，我们建议 从胃肠道和血液中采集VRE的人群水平进化动态研究 VRE-BSI，并鉴定促进VRE-BSI的细菌适应。我们的中心假设是VRE 从BSI分离出来的病毒具有基因适应能力，使它们能够在血液环境中生存。在目标1中， 我们将使用细菌种群水平的全基因组测序来识别与 VRE-BSI。我们建议从VRE胃肠道监测样本和VRE-BSI样本中收集匹配样本 大约150名患者，并对他们进行深入测序，以评估每个患者VRE群体的多样性 身体部位。我们还将比较随着时间的推移从持续性或慢性疾病患者那里收集的VRE-BSI群体 复发性VRE-BSI。我们将利用比较基因组学和基于选择的分析来识别细菌基因座 这些都是候选的选择目标。在目标2中，我们将量化VRE转录和突变的影响 翻译基因对抗生素的耐药性和耐受性。我们已经确定了具有适应性的候选人 RNA聚合酶亚基、核糖体蛋白、核糖体甲基转移酶和几个 转录调控因子。我们将调查：1)抗生素暴露与疾病发生之间的联系 在VRE-BSI患者的胃肠道和血液中的这些突变，2)这些突变对VRE的影响 转录和翻译，以及3)这些突变对抗性和/或耐受性的贡献。 用于治疗VRE-BSI的抗生素。在目标3中，我们将确定胶囊多糖中的突变 (CPS)和肠球菌多糖抗原(EPA)生物合成位点促进VRE生长和存活 BSI。我们将研究改变这些细胞表面相关多糖的突变对VRE的影响 在人的全血中存活，在人的中性粒细胞存在的情况下，以及在VRE的小鼠模型中 感染。总体而言，这项研究有可能改变我们对抗生素耐药性细菌如何 在人类感染期间适应。此外，筛选中的细菌基因和途径的鉴定 在VRE-BSI期间，将为开发针对抗生素耐药性的新治疗策略奠定基础 革兰氏阳性感染，死亡率高，给医疗保健系统带来巨大负担。

SUMMARY The long-term objective of this project is to understand how vancomycin-resistant enterococci (VRE) adapt during bloodstream infection (BSI) to better tolerate antibiotic and host immune defenses. Enterococci have evolved over hundreds of millions of years to colonize the gastrointestinal (GI) tract of animals, and they are well adapted to reside there. VRE causing BSI, however, face substantially different selective pressures, such as antibiotics in high concentrations, nutrient restriction, and host immune defenses. At our center over the past five years, patients with VRE-BSIs had a 30-day mortality rate of 36%, which was higher than BSIs due to all other ESKAPE pathogens. Additionally, VRE-BSIs are often difficult to treat, and nearly one third of patients with VRE- BSI experience either prolonged bacteremia (≥5 days), or recurrent infection within one year. Here we propose to study the population-level evolutionary dynamics of VRE sampled from the GI tract and blood of patients with VRE-BSI, and to characterize bacterial adaptations that promote VRE-BSI. Our central hypothesis is that VRE isolated from BSIs possess genetic adaptations that enable them to survive in the blood environment. In Aim 1, we will use bacterial population-level whole genome sequencing to identify genetic adaptations associated with VRE-BSI. We propose to collect matched samples from VRE GI tract surveillance specimens and VRE-BSI from approximately 150 patients, and to sequence them deeply to assess the diversity of the VRE population at each body site. We will also compare VRE-BSI populations collected over time from patients that have persistent or recurrent VRE-BSI. We will utilize comparative genomics and selection-based analyses to identify bacterial loci that are candidate targets for selection. In Aim 2, we will quantify the effect of mutations in VRE transcription and translation genes on antibiotic resistance and tolerance. We have already identified candidate adaptive mutations in RNA polymerase subunits, ribosomal proteins, a ribosome methyltransferase, and several transcriptional regulators. We will investigate: 1) The connection between antibiotic exposure and the occurrence of these mutations in the GI tract and blood of VRE-BSI patients, 2) The effects of these mutations on VRE transcription and translation, and 3) The contribution of these mutations to resistance and/or tolerance of antibiotics used to treat VRE-BSI. In Aim 3, we will determine whether mutations in the capsular polysaccharide (cps) and enterococcal polysaccharide antigen (epa) biosynthetic loci augment VRE growth and survival during BSI. We will investigate the impact of mutations that alter these cell surface-associated polysaccharides on VRE survival in whole human blood, in the presence of human neutrophils, as well as in a mouse model of VRE infection. Overall, this study has the potential to transform our understanding of how antibiotic-resistant bacteria adapt during human infection. In addition, the identification of bacterial genes and pathways under selection during VRE-BSI will lay the foundation for developing new therapeutic strategies that target antibiotic-resistant Gram-positive infections, which have high mortality and place a large burden on healthcare systems.