Viable but nonculturable Francisella tularensis is attenuated in a murine model 2300563

Abstract Introduction Francisella tularensis is a gram-negative intracellular bacterium that causes tularemia. F. tularensis LVS (a live vaccine attenuated surrogate strain) rapidly transitions to a viable but nonculturable state (VBNC) on media while maintaining viability and the ability to invade host cells. The role of VBNC in F. tularensis remains to be determined; however, in other pathogenic bacteria, it allows survival in hostile environments which would be experienced in nature or infections. Therefore, our study aimed to understand the effect of VBNC on pathogenesis in F. tularensis in a mammalian host model by determining whether cells remain VBNC or resuscitate to normal cellular activity. Methods To assess virulence and bacterial burden, BALB/c mice were intranasally challenged with varying doses of viable or VBNC LVS. At 21 days post challenge, tissues were homogenized and plated to determine CFU. We optimized flow cytometry protocols to quantify VBNC LVS in tissue samples to determine absolute cell counts by viability staining and immunolabeling. Results These experiments showed VBNC LVS remained attenuated in mice, displaying no morbidity or mortality for up to 21 days post-challenge. In contrast, mice challenged with viable LVS succumbed to challenge. Although VBNC LVS remained attenuated, bacteria were still detected in the lungs and spleen at the end of study based upon flow cytometry analysis. Additionally, mice challenged with VBNC LVS displayed no innate immune response from flow cytometry analysis of a myeloid cell panel. Surviving mice of the VBNC cohort were rechallenged with viable LVS or fully virulent Schu S4 to assess protection status. This revealed that VBNC exposure provided full protection against parental LVS re-challenge but afforded no protection against the Schu S4 strain. Conclusion Taken together, this work demonstrates VBNC LVS did not resuscitate upon entering a murine host by this challenge method. Furthermore, the VBNC state can provide protection against its parent strain. Funding Source DTRA-JSTO Topic Categories Microbial, Parasitic, and Fungal Immunology (MPF)