Aquatic long-term persistence ofFrancisella tularensisssp.holarcticais driven by water temperature and transition to a viable but non-culturable state

Francisella tularensis is a highly virulent bacterium causing tularemia zoonosis. An increasing proportion of infections occur through contaminated hydro-telluric sources, especially for the subspecies holarctica ( Fth ). Although this bacterium has been detected in several aquatic environments, the mechanisms of its long-term persistence in water are not yet elucidated. We evaluated the culturability and the viability of a virulent Fth strain in independent microcosms filled with nutrient-poor water. At 37°C, the bacteria remained culturable for only one week, while culturability was extended to 6 weeks at 18°C and up to 11 weeks at 4°C. However, while the viability of the bacteria declined similarly to culturability at 37°C, the viability of the bacteria remained stable overtime at 18°C and 4°C for more than 24 months, long after loss of culturability. We identified water temperature as one of the major factors driving the aquatic survival of Fth through a transition of the whole Fth population in a viable but non-culturable (VBNC) state. Low temperature of water (≤18°C) favors the persistence of the bacteria in a VBNC state, while a temperature above 30°C kills culturable and VBNC Fth bacteria. These findings provide new insights into the environmental cycle of Francisella tularensis that suggest that the yet unidentified primary reservoir of the subspecies holarctica may be the aquatic environment itself in which the bacteria could persist for months or years without the need for a host.