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

通过追踪宿主进化来确定导致持续性 MRSA 血流感染的机制

• 批准号: 10613457
• 负责人: MATTHEW J CULYBA
• 金额: $ 19.88万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-05-01 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10613457
• 关键词: Antibiotic Resistance; Antibiotics; Bacteremia; Biochemical Pathway; Biological; Biological Assay; Biology; Blood; Blood specimen; Cells; Citric Acid Cycle; Clinical; Complex; Data; Defect; Drug Interactions; Drug Modelings; Drug Tolerance; Environment; Evolution; Exposure to; Future; Genes; Genetic; Genetic Screening; Genetic screening method; Genome; Genotype; Growth; Hospitalization; Human; Immune; Immune Tolerance; Infection; Kinetics; Link; Macrophage; Measures; Metabolic Pathway; Metabolism; Methods; Modeling; Molecular; Mutate; Mutation; NADH; Pathogenesis; Pathway interactions; Patient-Focused Outcomes; Patients; Phagocytes; Phagosomes; Pharmacotherapy; Phenotype; Play; Population Sizes; Proteins; Reactive Oxygen Species; Resistance; Respiratory Burst; Role; Sepsis; Single Nucleotide Polymorphism; Staphylococcus aureus; Staphylococcus aureus infection; Stress; Testing; analytical tool; antibiotic tolerance; bacterial genome sequencing; chronic infection; design; effective therapy; experimental study; fitness; genomic locus; immune clearance; in vivo; innovation; insight; methicillin resistant Staphylococcus aureus; microbial; mortality; mutant; novel strategies; novel therapeutics; pathogen; pressure; respiratory; response; screening; trait

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.

项目摘要/摘要 由于甲氧西林抗葡萄球菌（MRSA）而引起的血液感染（BSI）携带约20％ 死亡率[1，2]。 [3]，PBSI的持续时间预测死亡率[2，12-14]。 PBSI [3]，强调耐受性是抗生素耐受性的重要原因。 特质与阻力不同，并且存在的特征是sturiers tos tos tos in Vivo受到了阻碍 了解临床环境中最重要的机制。 创新的基因筛查方法克服这些障碍。 不同的患者可以看作是微生物进化中自然发生的经验的生物学重复。 随着由于抗生素和免疫压力选择的细菌种群量崩溃，耐受突变体 将富集在同一遗传基因座中独立出现的突变 我们单独使用这种“基因型 - 曲线”方法的预预学研究，我们找到了证据 对于两种遗传途径的宿主进化，与抗生素耐受性密切相关。 在治疗MRSA-PBSI期间出现的突变体含有抗抗素和免疫的遗传适应。 宽容。我们建议通过以下特定AM识别和表征： AIM 1。确定MRSA-PBSI期间哪些基因演变与抗生素耐受性和 能量不平衡机制通常涉及新陈代谢的扰动 导致抗生素靶标的缓慢转移的速度缓慢[4，5]。 能量失调的冗余途径。 细胞的代谢网络中的节点和某些途径可能主导抗生素耐受性景观 将利用我们的遗传筛选来识别抗生素耐受性突变体并确定哪些基因 在MRSA-PBSI期间演变的与抗生素耐受性和能量失衡有关。 AIM 2。确定由于宿主病原体-Drugg而导致的MRSA-PBSI期间TCA周期缺陷是否演变 相互作用。 吞噬源性的活性氧形式的压力诱导金黄色葡萄球菌进入耐药态 通过减少三羧酸（TCA）周期的通量[6]。 在MRSA-PBSI期间演变的突变体 吞噬体，他们将在此设置中显示健身优势。 直接，通过执行我们感染吞噬细胞并测量存活和药物耐受性的实验。 这项研究不适用于理解持久性MRSA感染和TEE的基本生物学 在体内的基础机制和生物耐受性。