The Viable but Non-Culturable State of Francisella tularensis

Abstract

Tularemia is a zoonotic infection caused by the Category A biodefense agent Francisella tularensis.  This bacterium is maintained in small mammals such as rabbits but has also been isolated from the soil, natural water systems, and domestic water supplies.  Previous data has shown that F. tularensis can enter into a viable but non-culturable (VBNC) state where the bacterium cannot be cultivated by traditional methods, yet the bacterium is alive and maintains a measurable level of metabolic activity.  Since most conventional diagnostic tests depend upon cultivation of the bacteria, VBNC bacteria are a serious threat to public health.  Therefore, understanding how VBNC F. tularensis survive and persist in the environment and how they can be resuscitated back to an easily-detected, culturable form is of utmost clinical importance.  The focus of our project is determining culture conditions that cause F. tularensis to enter into the VBNC state in the laboratory setting.  We have tested various parameters including pH, temperature, and starvation as potential VBNC inducers. Culturability was measured by growth of F. tularensis on chocolate agar while viability was measured using a LIVE/DEAD fluorescent staining kit. To date, all conditions tested that have reduced the culturability of F. tularensis have also been associated with decreased viability. Current experimentation has focused on whether prolonged incubation of F. tularensis at mammalian body temperature will stimulate the bacteria to enter the VBNC state.  In the future, we plan to utilize laboratory-generated VBNC F.tularensis to determine which genes are expressed during this state that mediate survival.