Molecular Mechanisms of Pseudomonas aeruginosa Antibiotic Persistence in Monocultures and Microbial Communities

单一栽培和微生物群落中铜绿假单胞菌抗生素持久性的分子机制

• 批准号: 10749974
• 负责人: Patricia Jin Hare
• 金额: $ 4.45万
• 依托单位: UNIVERSITY OF CONNECTICUT SCH OF MED/DNT
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-01 至 2026-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10749974
• 关键词: Affect; Aftercare; Antibiotic Resistance; Antibiotic Therapy; Antibiotic susceptibility; Antibiotics; Automobile Driving; Bacteria; Bacterial Physiology; Basic Science; Biocide; Biological Assay; Cell Separation; Cell Survival; Cells; Chlorhexidine; Classification; Clinical; Coculture Techniques; Collaborations; Communities; Competitive Behavior; Complement; DNA Damage; DNA Repair Pathway; Dentists; Development; Dose; Drug Tolerance; Effectiveness; Engineering; Environment; Escherichia coli; Excision; Fellowship; Flow Cytometry; Fluoroquinolones; Future; Gene Expression; Genes; Genetic; Genetic Models; Genetic Transcription; Health; Health Care Costs; Heterogeneity; Individual; Infection; Knock-out; Knowledge; Laboratories; Lead; Learning; Levaquin; Lung infections; Membrane; Microbial Biofilms; Microscopy; Molecular; Nonhomologous DNA End Joining; Nutrient; Oral cavity; Pathway interactions; Pharmaceutical Preparations; Pharmacotherapy; Phenotype; Physiology; Population; Predisposition; Pseudomonas aeruginosa; Pseudomonas aeruginosa infection; Pulmonary Cystic Fibrosis; Recovery; Relapse; Reporter; Research; Research Proposals; Resistance; Resistance development; Resuscitation; Sampling; Scientist; Serial Passage; Site; Sorting; Specific qualifier value; Specificity; Sputum; Staphylococcus aureus; Surface; System; Technical Expertise; Techniques; Testing; Validation; antibiotic tolerance; antimicrobial; antimicrobial drug; bactericide; biological adaptation to stress; chronic infection; chronic wound; clinically relevant; design; differential expression; feeding; genetic testing; in vivo; infection management; insight; interest; knockout gene; microbial community; model organism; multidrug-resistant Pseudomonas aeruginosa; mutant; novel; opportunistic pathogen; oral bacteria; pathogenic bacteria; persistent bacteria; preservation; recurrent infection; repaired; resistance gene; response; secondary metabolite; segregation; skills; stress tolerance; stressor; tool; transcriptome sequencing; transcriptomics; treatment strategy

PROJECT SUMMARY Pseudomonas aeruginosa is an opportunistic pathogen that can withstand treatment with bactericidal antibiotics even when lacking identifiable resistance genes. It is thought that these recalcitrant infections are attributable to phenotypically antibiotic-tolerant cells called persisters. Despite the acknowledged contribution of P. aeruginosa to chronic and recurrent infections, there is a lack of basic research into cellular mechanisms that underlie P. aeruginosa antibiotic persistence. My central hypotheses in this research proposal are that the cellular responses following treatment will govern P. aeruginosa antibiotic persistence and resistance development (Aim 1), and that increased duration of coexistence with common co-isolate, S. aureus, will increase P. aeruginosa persistence by priming it in a more stress-tolerant state (Aim 2). This fellowship research will provide critical insight into persister physiology as well as the opportunity to learn cutting-edge techniques, analyses, and skills that will prepare me to lead independent research efforts in the future as a dentist-scientist interested in bacteria of the oral cavity. In Aim 1, I will investigate how P. aeruginosa persisters reawaken after drug treatment. I will perform RNA- seq to screen for genes that are differentially expressed between untreated cultures and cells that are viable after antibiotic treatment. Hits from this screen will be functionally validated by conducting persister assays with multiple genetic models for the genes of interest, including transcriptional reporters, knockout mutants, and inducible complementation strains. Significant genes of interest will be tested in biofilm cultures and in host- mimicking media to add clinical relevance. In Aim 2, I will determine how the duration of co-culture with S. aureus affects P. aeruginosa persistence and the physiologies of surviving cells. To efficiently passage and assay P. aeruginosa in co-culture, I am implementing a novel, dual-chambered apparatus that I designed, called the “H- Cell.” The H-Cell allows dynamic crosstalk between species while maintaining segregated populations for efficient sampling. I will determine the transcriptomic changes between P. aeruginosa persisters grown in monoculture or in H-Cell co-culture with S. aureus by RNA-seq. I will validate the hits by testing genetic constructs. Furthermore, I will test P. aeruginosa persistence in strains that are co-isolated with S. aureus from clinical sputum samples and thus have co-existed in a host environment. From my use of multiple bacterial strains, antimicrobials, and culture conditions, I aim to reveal the generalizability or specificity of P. aeruginosa persistence mechanisms and how they contribute to the development of antibiotic resistant progeny. Furthermore, I can apply the investigative approach, technical skills, and tools that I develop throughout this proposal to my future research on bacterial physiology in multispecies communities of the oral cavity. Altogether, completion of this research can inspire anti-persister strategies to reduce the burden of recalcitrant infections and their contributions to the broader antibiotic resistance crisis.

项目摘要 铜绿假单胞菌是一种条件致病菌，可以耐受杀菌剂的治疗。 抗生素，即使缺乏可识别的耐药基因。据认为，这些寄生虫感染是 这归因于被称为持留者的表型耐药性细胞。尽管公认的贡献， p.铜绿假单胞菌对慢性和复发性感染的作用，缺乏对细胞机制的基础研究， 是铜绿假单胞菌抗生素持久性的基础。在这项研究计划中，我的中心假设是， 治疗后的细胞反应将控制铜绿假单胞菌抗生素的持久性和耐药性 发展（目标1），并增加了与常见的共同分离，S。奥瑞乌斯 通过使铜绿假单胞菌处于更耐应激的状态来增加其持久性（目的2）。这项奖学金研究 将提供持续性生理学的关键见解，以及学习尖端技术的机会， 分析和技能，这将使我准备在未来作为一名牙医科学家领导独立的研究工作 对口腔细菌感兴趣。 在目标1中，我将研究铜绿假单胞菌如何在药物治疗后苏醒。我来做RNA- seq筛选未处理培养物和存活细胞之间差异表达的基因 抗生素治疗后。将通过进行持久性试验对该筛选的命中进行功能验证， 目的基因的多种遗传模型，包括转录报告基因、敲除突变体和 可诱导互补菌株。将在生物膜培养物和宿主中检测重要的感兴趣基因。 模仿媒体以增加临床相关性。在目标2中，我将确定与S.金黄色 影响铜绿假单胞菌的持久性和存活细胞的生理学。为了有效地传代和检测P. 铜绿假单胞菌在共培养中，我正在实施一种新的，我设计的双室装置，称为"H- 牢房。" H-Cell允许物种之间的动态串扰，同时保持隔离的种群， 高效采样我将确定铜绿假单胞菌持续生长的转录组变化， 单培养或在H细胞与S.通过RNA-seq.我将通过基因测试来验证命中 结构。此外，我将检测铜绿假单胞菌在与S.金黄色 临床痰样品，并因此在宿主环境中共存。 从我使用的多种细菌菌株，抗菌剂和培养条件，我的目的是揭示 铜绿假单胞菌持久性机制的普遍性或特异性，以及它们如何有助于 产生抗生素抗性后代。此外，我可以运用调查方法，技术技能， 以及我在这个提案中开发的工具，用于我未来对多物种细菌生理学的研究， 口腔的社区。总之，这项研究的完成可以激发抗持久性策略， 减少耐药性感染的负担及其对更广泛的抗生素耐药性危机的贡献。