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.

项目概要 铜绿假单胞菌是一种机会致病菌，可以耐受杀菌剂的治疗 即使缺乏可识别的耐药基因，人们也认为这些顽固性感染是抗生素。 尽管公认的贡献是由称为持久细胞的表型抗生素耐受细胞引起的。 铜绿假单胞菌对慢性和反复感染的细胞机制缺乏基础研究 铜绿假单胞菌抗生素持久性的基础是我在本研究提案中的中心假设是： 治疗后的细胞反应将决定铜绿假单胞菌抗生素的持久性和耐药性 发展（目标 1），并且增加与常见共分离株金黄色葡萄球菌共存的持续时间，将 通过将铜绿假单胞菌置于更耐压的状态来提高其持久性（目标 2）。 将提供对持久生理学的批判性见解以及学习尖端技术的机会， 分析和技能将使我准备好在未来作为牙医科学家领导独立研究工作 对口腔细菌感兴趣。 在目标 1 中，我将研究铜绿假单胞菌在药物治疗后如何重新苏醒，我将进行 RNA-。 seq 筛选未处理的培养物和存活细胞之间差异表达的基因 抗生素治疗后，将通过进行持久化分析来对来自该屏幕的命中进行功能验证。 感兴趣的基因的多种遗传模型，包括转录产生者、敲除突变体和 将在生物膜培养物和宿主中测试重要的感兴趣基因。 模拟培养基以增加临床相关性 在目标 2 中，我将确定与金黄色葡萄球菌共培养的持续时间。 影响铜绿假单胞菌的持久性和存活细胞的生理学，以有效传代和 P 测定。 在共培养铜绿假单胞菌中，我正在实施一种我设计的新颖的双室装置，称为“H- H-Cell 允许物种之间动态串扰，同时保持种群隔离 我将确定生长的铜绿假单胞菌之间的转录组变化。 通过 RNA-seq 进行单一培养或 H-Cell 与金黄色葡萄球菌共培养，我将通过测试遗传来验证命中结果。 此外，我将测试与金黄色葡萄球菌共同分离的菌株中铜绿假单胞菌的持久性。 临床痰标本，因此在宿主环境中共存。 通过我对多种细菌菌株、抗菌剂和培养条件的使用，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促进 此外，我可以应用研究方法、技术技能， 以及我在整个提案中为我未来的多物种细菌生理学研究开发的工具 总而言之，这项研究的完成可以激发反持久性策略。 减少顽固性感染的负担及其对更广泛的抗生素耐药性危机的影响。