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

, the 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 forms biofilms in aquatic environments. We utilized two fully virulent wild-type strains: FSC200 ( subsp. ) and Schu S4 ( subsp. ) and three control strains, the attenuated live vaccine strain (LVS; subsp. ), a Schu S4 Δ mutant that is documented to form biofilms, and the low-virulence strain U112 of the closely related species 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 Δ formed biofilms, but strains FSC200 and Schu S4 and the 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 1- and 2-fold log reductions, 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 subsp. in conditions that mimic overwinter survival in aquatic environments. Tularemia, a disease caused by the environmental bacterium , is characterized by acute febrile illness. 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 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 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 may persist long-term in the environment.