Determining the mechanisms that cause persistent MRSA bloodstream infection by tracking in-host evolution

项目摘要/摘要 耐甲氧西林金黄色葡萄球菌(MRSA)引起的血流感染(BSI)约占20% 死亡率[1，2]。MSRA对抗生素杀灭表现出耐受性[11]，有导致持续性BSI(PBSI)的倾向 [3]，PBSI的持续时间预测死亡率[2，12-14]。耐甲氧西林金黄色葡萄球菌很少获得坦率的抗生素耐药性 PBSI[3]，强调耐受性是患者预后差的一个重要原因。抗生素耐药性是一个复杂的问题 这与抗药性是不同的，在体内研究它有很大的障碍，阻碍了 了解临床环境中最重要的机制的进展。在这项提案中，我们提出了一个 创新的基因筛查方法克服了这些障碍。发生在以下地区的MRSA-PBSI发作 不同的患者可以被视为微生物进化中自然发生的实验的生物复制。 由于抗生素和免疫压力的选择，细菌种群数量急剧减少，耐受性突变体 会变得更加丰富。在同一基因座上以超过偶然率的速度独立出现的突变 在生物学上有意义。在初步研究中，使用这种“基因优先”的方法，我们发现了证据 与抗生素耐受性密切相关的两条遗传途径的宿主内进化。我们的中心假设是 在治疗MRSA-PBSI过程中出现的突变株包含对抗生素和免疫的遗传适应 宽容。我们建议通过以下具体目标来确定和描述这些途径： 目的1.确定在MRSA-PBSI期间进化的哪些基因与抗生素耐受性和 能源失衡。耐受机制常常涉及新陈代谢的紊乱，导致“低能量”。 导致抗生素靶标周转缓慢的状态[4，5]。这样的扰动可能通过各种不同的 汇聚在能源失调上的多余路径。或者，体内条件可能会产生特定的压力。 细胞代谢网络中的节点和一些途径可能主导着抗生素耐受性的格局。我们 将利用我们的基因筛选方法来识别耐药突变并确定哪些基因 在MRSA-PBSI期间的演变与抗生素耐受性和能量失衡有关。 目的2.确定在MRSA-PBSI过程中是否由于宿主-病原体-药物而发生TCA循环缺陷 互动。恶劣的环境可以诱导抗生素耐受性，一个主要的模式是宿主免疫 压力以吞噬细胞衍生的活性氧的形式诱导金黄色葡萄球菌进入耐药状态 通过三羧酸(TCA)循环减少通量[6]。在我们的初步数据中，我们确定了TCA循环 在MRSA-PBSI期间进化的突变体。如果这些突变体是通过与野生型MRSA竞争而进化的 吞噬小体，它们在这种情况下会显示出健身优势。我们将利用这些突变体来测试这个模型 直接，通过进行实验，我们感染吞噬细胞，测量存活率和药物耐受性。 这项研究对于了解耐甲氧西林金黄色葡萄球菌持续感染的基础生物学和 体内抗生素耐受的潜在机制。这些信息将为新疗法的设计提供信息。

Project Summary/Abstract Bloodstream infection (BSI) due to methicillin-resistant Staphylococcus aureus (MRSA) carries ~20% mortality [1, 2]. MSRA displays tolerance to antibiotic killing [11], has a propensity to cause persistent BSI (pBSI) [3], and the duration of pBSI predicts mortality [2, 12-14]. MRSA rarely acquires frank antibiotic resistance during pBSI [3], highlighting tolerance as an important cause of poor patient outcomes. Antibiotic tolerance is a complex trait, which is distinct from resistance, and there are significant barriers to its study in vivo that have hampered progress on understanding the most important mechanisms in clinical settings. In this proposal, we advance an innovative genetic screening approach to overcome these barriers. Episodes of MRSA-pBSI that occur in different patients can be viewed as biological replicates of a naturally occurring experiment in microbial evolution. As bacterial population sizes collapse due to selection from antibiotic and immune pressure, tolerant mutants will become enriched. Mutations that arise independently in the same genetic loci at a rate that exceed chance alone, are biologically meaningful. In preliminary studies, using this “genotype-first” approach, we found evidence for in-host evolution of two genetic pathways strongly linked to antibiotic tolerance. Our central hypothesis is that mutants that arise during the treatment of MRSA-pBSI contain genetic adaptations for antibiotic and immune tolerance. We propose to identify and characterize these pathways through the following specific aims: Aim 1. Determine which genes evolving during MRSA-pBSI are associated with antibiotic tolerance and energy imbalance. Tolerance mechanisms often involve perturbations in metabolism, causing a ‘low energy’ state that leads to slow turnover of antibiotic targets [4, 5]. Such perturbations could arise through a variety of redundant pathways that converge on energy dysregulation. Alternatively, in vivo conditions may stress specific nodes in the cell’s metabolic networks and some pathways may dominate the antibiotic tolerance landscape. We will utilize our genetic screening approach to identify antibiotic tolerant mutants and determine which genes evolving during MRSA-pBSI are associated with antibiotic tolerance and energy imbalance. Aim 2. Determine if TCA cycle defects evolve during MRSA-pBSI due to a host-pathogen-drug interaction. Antibiotic tolerance can be induced by harsh environments and a leading model is that host immune pressure in the form of phagocyte-derived reactive oxygen species induces S. aureus into a drug-tolerant state by reducing flux through the tricarboxylic acid (TCA) cycle [6]. In our preliminary data, we identified TCA cycle mutants that evolved during MRSA-pBSI. If these mutants evolved by outcompeting wild-type MRSA in phagosomes, they will display a fitness advantage in this setting. We will utilize these mutants to test this model directly, by performing experiments where we infect phagocytes and measure survival and drug tolerance. This study is important for understanding the fundamental biology of persistent MRSA infection and the mechanisms underlying antibiotic tolerance in vivo. This information will inform the design of novel therapies.