Aminoglycoside-Enabled Elucidation of Persister Metabolism

氨基糖苷类药物对持续代谢的阐明

• 批准号: 8605521
• 负责人: Mark P Brynildsen
• 金额: $ 22.84万
• 依托单位: PRINCETON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-02-01 至 2016-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8605521
• 关键词: Accounting; Affect; Algorithms; Aminoglycosides; Ampicillin; Antibiotic Therapy; Antibiotics; Back; Bacteria; Biological Assay; Biology; Carbon; Cells; Characteristics; Chemosensitization; Chronic; Communicable Diseases; Computational Technique; Computing Methodologies; Consumption; Cytochromes; Data; Development; Enzymes; Escherichia coli; Event; Film; Future; Generations; Goals; Growth; Heterogeneity; Hospitals; Infection; Kanamycin; Knowledge; Lead; Maintenance; Measures; Metabolic; Metabolic Pathway; Metabolism; Methods; Microbial Biofilms; Microbiology; Minor; Nutrient; Ofloxacin; Outcome; Pathway interactions; Phase; Phenotype; Physiology; Population; Pseudomonas aeruginosa; Reaction; Recurrence; Relapse; Research; Respiration; Sampling; Source; Staphylococcus aureus; Stress; Survivors; Techniques; Testing; Therapeutic Intervention; Work; antimicrobial; effective therapy; feeding; improved; killings; metabolic abnormality assessment; novel therapeutics; pathogen; public health relevance; research study

DESCRIPTION (provided by applicant): Bacterial persisters tolerate antibiotic treatment, and underlie the propensity of biofilm infections to relapse. An improved understanding of persister physiology will lead to the development of more effective therapies against biofilm-utilizing pathogens such as Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. Persister metabolism is of particular importance, as it influences entry into, maintenance of, and exit from this antibiotic-tolerant state. Unfortunately, persisters are a minor, transient subpopulation whose physiology is easily obscured by more abundant phenotypes (e.g., viable but non-culturable cells (VBNCs)). Recent evidence suggests that current persister isolation techniques provide samples with many more VBNCs than persisters. Without improved isolation techniques, the distinguishing feature between VBNCs and persisters, growth- resumption on standard media, must be used to characterize persister physiology. Here we propose to develop a method to elucidate the metabolic abilities of persisters from survival data, and thus circumvent the present isolation difficulties. Recent work has demonstrated that persisters can catabolize carbon sources, remain non-replicative, and yet become susceptible to aminoglycosides. Here we propose to harness this phenomenon to chart the metabolic capabilities of persisters. To accomplish this goal, we will develop a rapid AG potentiation assay, develop a computational approach to analyze the resulting survival data and direct further experimentation, and finally, demonstrate the utility of our approach by charting the metabolic abilities of three persister populations. To develop a rapid AG potentiation assay, we will use phenotype arrays (each well contains a different nutrient) to simultaneously measure AG potentiation and the necessary controls from hundreds of separate nutrients. To analyze the resulting survival data, we will use mixed integer linear optimization to generate an ensemble of non-redundant minimal metabolic pathways capable of explaining the data. These pathways will be clustered, and the reactions that most significantly discriminate between competing clusters will be experimentally perturbed to determine the metabolic capabilities of persisters. To demonstrate the utility of our approach, we will use our technique to study the metabolic abilities of three persister populations: exponential phase persisters tolerant to ofloxacin, exponential phase persisters tolerant to ampicillin, and stationary persisters tolerant to ofloxacin. Results from this proposal will fill fundamental knowledge gaps in persister metabolism, identify new avenues for therapeutic intervention through disruption of persister maintenance or enhancement of persister awakening, and impact the fields of network biology, microbiology, and infectious disease.

描述(由申请人提供)：细菌持续者耐受抗生素治疗，并导致生物膜感染复发的倾向。对持续生理学的了解将导致开发更有效的治疗方法来对抗利用生物被膜的病原体，如大肠埃希菌、铜绿假单胞菌和金黄色葡萄球菌。持久代谢特别重要，因为它影响进入、维持和退出这种抗生素耐受状态。不幸的是，持久者是一个较小的、短暂的亚群，他们的生理很容易被更丰富的表型(例如，活的但不可培养的细胞(VBNC))所掩盖。最近的证据表明，目前的持久分离技术提供的样本比持久分离的VBNC要多得多。如果没有改进的分离技术，必须使用VBNC和持久体之间的区别特征，即在标准培养基上的生长恢复，来表征持久生理学。在这里，我们建议开发一种方法来从生存数据中阐明持久者的代谢能力，从而绕过目前的隔离困难。最近的研究表明，持久者可以分解碳源，保持非复制性，但对氨基糖苷类药物敏感。在这里，我们建议利用这一现象来描绘持久者的新陈代谢能力。为了实现这一目标，我们将开发一种快速的AG增强分析，开发一种计算方法来分析所产生的生存数据并指导进一步的实验，最后，通过绘制三个外围种群的代谢能力图来展示我们方法的有效性。为了开发一种快速的AG增强分析，我们将使用表型阵列(每个井包含不同的营养物质)来同时测量AG增强作用和数百种不同营养物质的必要对照。为了分析最终的生存数据，我们将使用混合整数线性优化来生成能够解释数据的非冗余最小代谢路径集合。这些途径将被聚集在一起，最显著区分竞争簇的反应将在实验上受到干扰，以确定持久者的代谢能力。为了证明我们方法的实用性，我们将使用我们的技术来研究新陈代谢能力 对氧氟沙星耐受性的指数期持续者，对氨苄西林耐受性的指数期持续者，以及耐氧氟沙星的稳定期持续者。这项提议的结果将填补持续性新陈代谢的基本知识空白，通过扰乱持久性维持或增强持久性唤醒来确定治疗干预的新途径，并影响网络生物学、微生物学和传染病领域。