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

Abstract

Francisella tularensis, 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 F. tularensis forms biofilms in aquatic environments. We utilized two fully virulent wild-type strains: FSC200 (Francisella tularensis subsp. holarctica) and Schu S4 (Francisella 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 strain U112 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 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 F. tularensis subsp. holarctica in conditions that mimic overwinter 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.

Keywords: Francisella tularensis; aquatic environment; biofilms; long-term persistence; tularemia.

FIG 1

Biofilm formation of Francisella strains at 20°C (left) and 4°C (right) measured over time. Bacterial strains were incubated in a saline solution (0.9% NaCl), and biofilm formation was quantified using crystal violet stain and optical density measurement performed at 2, 3, 4, 6, 8, 10, 12, and 24 weeks incubation. Box plots present the median, quartile, minimum, and maximum of measurements. All data from the 2-, 3-, 4-, 6-, 8-, 10-, 12-, and 24-week measurements are presented in one whisker box plot per strain.

FIG 2

Confocal laser scanning microscopy of U112pKK289Kan-gfp (A) and LVSpKK289Kan-gfp (B), expressing GFP, after 1 week in 0.9% NaCl in 20°C.

FIG 3

Viable counts of planktonic bacteria in the biofilm assay over time (0, 12, 14, 20, and 24 weeks). Francisella strains were incubated in 0.9% NaCl at 20°C and 4°C. Columns show means for six replicates (except those for FSC200 and the LVS at 24 weeks, which show means for three replicates), and error bars represent standard deviations. Absence of bars indicates no CFU.

FIG 4

Virulence of Francisella tularensis FSC200 (3 mice) and Schu S4 (3 mice) populations, incubated at 4°C for 24 weeks, in the C57BL/6J mouse model. FSC200 and Schu S4 from overnight cultures and an infectious dose of 10² CFU were used as a control (3 mice for each strain) and 0.9% NaCl was used as a negative control (2 mice).