Long-Term Survival of Virulent Tularemia Pathogens outside a Host in Conditions That Mimic Natural Aquatic Environments

Abstract

Francisella tularensis, causative agent of the zoonotic disease tularemia, can cause seasonal outbreaks of acute febrile illness in humans with disease peaks in late summer to autumn. Interestingly, its mechanisms for environmental persistence between outbreaks are poorly understood. One hypothesis is that F. tularensis forms biofilm in aquatic environments. We utilized two fully virulent wild type strains: FSC200 (F. tularensis subsp. holarctica) and Schu S4 (F. tularensis subsp. tularensis); and three control strains: the attenuated Live Vaccine Strain (LVS; F. tularensis subsp. holarctica), a Schu S4 ΔwbtI mutant that is documented to form biofilms, and the low virulence U112 strain of the closely-related species Francisella novicida. Strains were incubated in saline solution (0.9% NaCl) microcosms for 24 weeks at both 4°C and 20°C whereupon viability and biofilm formation were measured. These temperatures were selected to approximate winter and summer temperatures of fresh water in Scandinavia, respectively. U112 and Schu S4 ΔwbtI formed biofilms, but F. tularensis strains FSC200, Schu S4, and LVS did not. All strains exhibited prolonged viability at 4°C compared to 20°C. U112 and FSC200 displayed remarkable long-term persistence at 4°C, with only a one- and two-fold log reduction, respectively, of viable cells after 24 weeks. Schu S4 exhibited lower survival, yielding no viable cells by week 20. At 24 weeks, cells from FSC200, but not from Schu S4, were still fully virulent in mice. Taken together, these results demonstrate biofilm-independent, long-term survival of pathogenic F. tularensis subsp. holarctica in conditions that mimic over-winter survival in aquatic environments. Importance Tularemia, a disease caused by the environmental bacterium, Francisella tularensis, is characterized by acute febrile illness. F. tularensis is highly infectious: as few as 10 organisms can cause human disease. Tularemia is not known to be spread from person to person. Rather, all human infections are independently acquired from the environment via the bite of blood-feeding arthropods, ingestion of infected food or water, or inhalation of aerosolized bacteria. Despite the environmental origins of human disease events, the ecological factors governing the long-term persistence of F. tularensis in nature between seasonal human outbreaks are poorly understood. The significance of our research is in identifying conditions that promote long-term survival of fully virulent F. tularensis outside a mammalian host or insect vector. These conditions are similar to those found in natural aquatic environments in winter and provide important new insights on how F. tularensis may persist long-term in the environment.

Presence of Francisella tularensis subsp. holarctica DNA in the Aquatic Environment in France

“…holarctica in surface water raises the question of how this bacterium can survive and persist in such aquatic environments and why it does not infect thousands of people, according to the high virulence of this bacterium. Experimental studies have shown that F. tularensis can survive in water microcosms for months [ 26 , 27 , 28 , 29 ], firstly in a culturable form and then in a potentially viable but nonculturable form (VBNC) [ 26 , 28 ]. Long-term planktonic forms of F. tularensis subsp.…”

“…holarctica have been found to be still virulent in mice, whereas similar forms of F. tularensis subsp. tularensis did not [ 29 ]. VBNC forms of F. tularensis subsp.…”

“…However, no correlation was found between the presence of Acanthamoeba and Francisella DNA in water samples. There are conflicting studies about the capacity of F. tularensis to produce biofilms to survive in water [ 29 , 35 ]. Finally, this bacterium was shown to be able to survive within mosquito larvae [ 36 ].…”

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

“…Recently while studying biofilm formation of F. tularensis in aquatic environments, Golovliov et al identified that Fth remained culturable and infectious in a mice model after 24 weeks of incubation at 4°C in low nutrient water containing 9 g/L of NaCl. They suggested that this improved survival at low temperature in freshwater may be a critical mechanism to help the bacteria overwinter and survive between hostassociated replication events [4]. In such situations the bacteria may choose to switch to a dormancy state that reduces competition with actively growing cells.…”

Francisella novicida can utilize Paramecium bursaria as its potential host

“…However, the ecology of F. novicida in the natural environment and the route of infection for humans remain unclear. Many of the characteristics of F. novicida , such as its biofilm‐forming ability, long‐term viability in water and establishment of relationships with aquatic protists such as intra‐vacuolar proliferation within Hartmannella vermiformis , suggest the possibility of its stable survival in the aquatic environment (Santic et al ., 2011; Siebert et al ., 2020; Golovliov et al ., 2021). Based on these facts, it is reasonable to consider F. novicida infection a water‐borne disease.…”