Identifying the pathways associated with bacterial antibiotic persistence within host tissues

PROJECT SUMMARY Residual subpopulations of antibiotic-susceptible bacteria can remain within host tissues following antibiotic treatment. These surviving bacteria are called persister cells, which are transiently tolerant to high levels of antibiotic, and can cause serious relapsing infection after treatment. Critically, current treatment strategies do not target persisters. To fully eradicate all bacterial cells, treatments are prolonged, increasing patient and clinical costs. Prolonged antibiotic exposure can promote antibiotic resistance, further emphasizing the need to improve treatment efficacy. Improved treatment strategies would simultaneously target all members of the bacterial population, including persisters. However, persisters have been primarily studied in culture, and relevant persister cell-specific drug targets within host tissues are largely undefined. Bacteria behave very differently in host tissues, where nutrient limitation and antimicrobial host defenses activate strong stress response pathways in bacterial pathogens. We predict persisters utilize distinct, potentially novel, survival strategies within the host environment. To study bacterial antibiotic persistence within host tissues, we established a mouse model of doxycycline treatment of Yersinia pseudotuberculosis splenic deep tissue infection. Doxycycline is an effective treatment for human Yersinia infection, but requires 7 days continuous treatment, which has been incorporated into our mouse model. Prior to antibiotic treatment, Y. pseudotuberculosis replicate to form clusters of extracellular bacteria that directly interface with a layer of neutrophils that are, in turn, enveloped by a layer of monocytes. In the initial 4h of doxycycline treatment, we observe a significant decrease in viable bacterial numbers, which correlates with a wave of neutrophil infiltration into the spleen. However, a residual bacterial subpopulation (~10%) remain in the spleen throughout the 7-day treatment. Bacterial cells resume growth and cause lethality when antibiotic concentrations wane, defining these cells as persisters. We hypothesize that interactions with neutrophils and monocytes predispose persisters to survive antibiotic treatment, and prolonged antibiotic exposure promotes additional transcriptional and genetic changes within persister cells. Utilizing our fluorescent reporter system to detect viable, doxycycline-exposed bacteria within the mouse spleen, we will: 1) identify the transcriptional, proteomic, and genetic changes specific to surviving bacteria within antibiotic-treated mice, 2) determine whether specific bacterial targets are critical for antibiotic persistence in the host, and 3) determine if monocyte or neutrophil interactions promote antibiotic persistence. We hypothesize activated neutrophils initially reduce the bacterial burden, and we will determine if evasion of neutrophil-mediated killing promotes persister cell survival. Identifying persister cell survival strategies within host tissues will provide critical information to advance the field and enable the development of more efficacious therapeutic strategies against bacterial infections.