Mechanisms of antibiotic persistence in a mouse model of systemic infection

PROJECT SUMMARY Systemic bacterial infections are a major cause of morbidity and mortality globally, and antibiotic treatment failures constitute a major global health issue. In recent years, there has been increasing attention to previously underappreciated mechanisms of antibiotic treatment failure, specifically the role of phenotypically distinct bacterial subpopulations which fail to be eliminated. During an infection, individual bacterial cells are differentially exposed to a wide range of host-derived stressors. This can result in the emergence of phenotypically distinct subpopulations with reduced susceptibility to antibiotics – a phenomenon termed antibiotic persistence. Antibiotic persistence poses a significant therapeutic challenge; surviving bacterial subpopulations can promote long-term and relapsing infections. The development of new therapeutic strategies for antibiotic persistent infections is currently limited by major gaps in our understanding of the mechanisms responsible for the development and maintenance of antibiotic persistence in the context of a mammalian infection. Our laboratory has developed a mouse model for prolonged antibiotic treatment of a systemic bacterial infection, as a system for studying antibiotic persistence within the host environment. Mice are inoculated intravenously (i.v.) with the Gram-negative bacterium Yersinia pseudotuberculosis (Yptb). Yptb is a natural pathogen of humans and rodents, and there are many genetic tools well-established in Yptb. Upon i.v. inoculation, the bacteria disseminate to deep tissue sites (primarily the spleen), where they form structures referred to as microcolonies by 48 hours post-infection. Microcolonies consist of a bacterial center surrounded by host immune cells (neutrophils and monocytes/macrophages). Bacterial centers within each microcolony are complex and dynamic populations, with distinct bacterial subpopulations, making them a powerful model for studying phenotypic heterogeneity. The central hypothesis of this project is that exposure to host- derived stressors will induce persistent bacterial phenotypes, which will be further induced and maintained during the course of antibiotic treatment, until antibiotic levels wane. Here we will use a new bacterial RNA sequencing approach, along with existing genetic tools to study antibiotic persistence mechanisms in our newly developed prolonged treatment mouse model. Through this project we hope to provide mechanistic insight to inform the development of new treatment strategies, including combination therapies to specifically target multiple populations.