Adaptation of vancomycin-resistant enterococci during bloodstream infection

总结 该项目的长期目标是了解万古霉素耐药肠球菌（VRE）如何适应 在血液感染（BSI）期间，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，它们在动物的胃肠道（GI）中定居，并且它们很好地 适合居住在那里。然而，引起BSI的VRE面临着基本上不同的选择性压力，例如 高浓度抗生素、营养限制和宿主免疫防御。在过去的五年里， 在2010年，VRE-BSI患者的30天死亡率为36%，高于所有其他原因导致的BSI。 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经历长期菌血症（≥5天）或在一年内复发感染。在这里我们建议 研究从胃肠道和血液中取样的VRE的群体水平进化动力学， VRE-BSI，并表征促进VRE-BSI的细菌适应。我们的中心假设是， 从BSIs中分离出来的细菌具有遗传适应性，使它们能够在血液环境中生存。在目标1中， 我们将使用细菌群体水平的全基因组测序来识别与以下疾病相关的遗传适应性： VRE-BSI。我们建议从VRE胃肠道监测标本和VRE-BSI中采集匹配样本， 大约150名患者，并对其进行深度测序，以评估每个阶段VRE人群的多样性。 身体部位。我们还将比较一段时间内从持续性或非持续性癫痫患者中收集的VRE-BSI人群， 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确定细菌位点 作为选择的候选目标。在目标2中，我们将量化VRE转录中突变的影响， 翻译基因对抗生素耐药性和耐受性的影响。我们已经确定了候选自适应 RNA聚合酶亚基、核糖体蛋白、核糖体甲基转移酶和几种 转录调节因子。我们将调查：1）抗生素暴露与发生之间的联系 VRE-BSI患者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）这些突变对耐药性和/或耐受性的贡献。 用于治疗VRE-BSI的抗生素。在目标3中，我们将确定是否在荚膜多糖突变， (cps)和肠球菌多糖抗原（epa）生物合成位点增加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。 在人全血中、存在人中性粒细胞时以及在VRE小鼠模型中的存活率 感染总的来说，这项研究有可能改变我们对抗药性细菌如何在体内生长的理解。 适应人类感染。此外，在选择下鉴定细菌基因和途径 在VRE-BSI期间，将为开发新的治疗策略奠定基础， 革兰氏阳性感染死亡率高，给医疗保健系统带来沉重负担。

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.