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

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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.