PhD defense of  Camille Brunet from TIMC TrEE on march 2022, the 31th at 2pm:

« Modes and mechanisms of environmental survival
of the bacterium Francisella tularensis ssp. holarctica »
 

• Place : Salle Mazaré, Bâtiment Boucherle, 23 avenue des Maquis du Grésivaudan, 38700 La Tronche
   

Jury :

• Prof. Patrice Morand, UGA, President
• Prof. Nadia Haddad, Ecole vétérinaire d'Alfort, Reporter
• Prof. Sophie Jarraud, Université de Lyon, Reporter
• Dr. Thomas Henry, Université de Lyon, Examiner
• Dr. Emmanuelle Guillot-Combe, Direction Générale de l'Armement, Invited
• Prof. Max Maurin, UGA, Supervisor
• Dr. Yvan Caspar, UGA, Co-supervisor

  Keywords:  

Francisella tularensis, tularemia, viable but non-culturable bacteria, aquatic reservoir, environmental persistence
 

Abstract:

Tularemia is a zoonosis caused by the Gram-negative bacterium Francisella tularensis. Two subspecies are virulent to humans: F. tularensis ssp. tularensis, found only in North America, and F. tularensis ssp. holarctica, found throughout the Northern Hemisphere and in Australia. In particular, it is possible to be contaminated by aerosols containing the bacterium, which causes pulmonary infections. This possibility, as well as the high pathogenicity of this bacterium for humans, classifies it as a potential bioterrorism agent. In order to control an accidental spread of F. tularensis, it is necessary to understand its survival mechanisms in the environment. The terrestrial life cycle of F. tularensis ssp. holarctica is relatively well characterized. In contrast, the aquatic life cycle of this subspecies is still poorly characterized. Sporadic human infections and epidemics of aquatic origin occur after aquatic activities, ingestion of contaminated water or mosquito bites. A few studies have reported the persistence of F. tularensis for several weeks in the environment, without identifying the mechanisms of this survival.

The objective of this thesis work was to characterize the persistence of F. tularensis ssp. holarctica in an aquatic environment. Two field studies conducted in 2019 and 2020 demonstrated the presence of F. tularensis DNA in environmental bodies of water in France. Across both campaigns, 9.2% of the 87 water samples analyzed contained DNA from the tularemia-causing bacterium. Although the presence of DNA is not a proof of aquatic persistence of the bacterium in an infectious form, this work questions the survival of F. tularensis in environmental water, and its responsibility in some cases of tularemia.

In vitro models of environmental survival of a virulent strain of F. tularensis ssp. holarctica demonstrated that this bacterium was able to enter a dormant state called the viable but non-cultivable (VBNC) state. In this state, the bacteria were no longer able to grows in a suitable rich environment, but remained alive. This state allowed the bacteria to persist for more than 18 months in the water, evading conventional detection tools based on bacterial culture. Water temperature appears to be a critical factor for the survival of F. tularensis, as the VBNC state was only induced in cool water (4 and 18°C), whereas warmer water (37°C) was lethal to the bacteria within a few days. Although the VBNC state is a means of resisting environmental stresses, bacteria in the VBNC state remained temperature sensitive, which may explain the geographic distribution of tularemia and bacterial persistence limited to Northern Hemisphere countries.

A proteomic characterization of F. tularensis in the VBNC state was initiated to finish this thesis work. Remodeling of the bacterial envelope and regulation of virulence factors seems to take place in this dormant state. A reduction of proteins synthesizing peptidoglycan, type IV pili, type VI secretion system and O-antigen was observed.

The complementary results of this work allowed us to propose an update of the aquatic life cycle of F. tularensis ssp. holarctica. This thesis work demonstrates that F. tularensis ssp. holarctica is capable of surviving several months in an aquatic environment in the VBNC state raising new questions about the resistance and virulence of these bacteria in the VBNC state in the environment that will need to be investigated in the future.