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

项目总结 铜绿假单胞菌是一种能耐受杀菌治疗的条件致病菌。 抗生素，即使在缺乏可识别的耐药性基因时也是如此。据认为，这些顽固性感染是 可归因于表型耐受抗生素的细胞称为持续体。尽管公认的贡献是 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。 铜绿假单胞菌抗生素持久性的基础。我在这项研究提案中的中心假设是 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)，与共同共同分离物金黄色葡萄球菌共存时间延长将 通过使铜绿假单胞菌处于更耐受压力的状态来增加其持久性(目标2)。这项奖学金研究 将提供对持久生理学的重要见解以及学习尖端技术的机会， 分析和技能，这将使我做好准备，将来作为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是如何在药物治疗后重新苏醒的。我会做核糖核酸- SEQ用于筛选未经处理的培养细胞和存活细胞之间差异表达的基因 在抗生素治疗后。来自此屏幕的点击将通过执行持久分析来进行功能验证 感兴趣基因的多种遗传模型，包括转录报告、敲除突变体和 可诱导的互补菌株。重要的基因将在生物膜培养和宿主中进行测试- 模仿媒体以增加临床相关性。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。为了有效地传代和检测P. 铜绿假单胞菌在共培养中，我正在实施一种我设计的新颖的双腔装置，名为“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久菌在 单独培养或H细胞与金黄色葡萄球菌共培养。我会通过检测基因来确认命中结果 构造。此外，我将在与金黄色葡萄球菌共分离的菌株中测试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，我的目标是揭示 铜绿假单胞菌持久性机制的概括性或特异性及其对 抗生素耐药后代的发育。此外，我可以运用调查方法，技术技能， 以及我在整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启发反持久者策略 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贡献。

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.