A slow decay to extinction: cold temperatures and reduced microbial complexity prolong the persistence of planktonic Francisella tularensis

ABSTRACT Francisella tularensis is a zoonotic pathogen transmitted to humans through contact with infected animals, arthropod vectors, or contaminated environmental sources. Although replication occurs primarily within eukaryotic host cells, persistence outside a host is thought to be critical for long-term maintenance and transmission in natural ecosystems. To better understand persistence in aquatic habitats outside of mammalian hosts, we studied virulent strains of F. tularensis subspecies tularensis (type A) and subspecies holarctica (type B) incubated in a planktonic state (free living in water) under various axenic aquatic conditions. After 1 year in cold, nutrient-poor treatments, the type B strain (FSC200) caused disease in mice and remained culturable on laboratory media, whereas the type A strain (Schu S4) caused disease but with delayed onset and became non-responsive to laboratory growth conditions. In 1.5-year persistence experiments, type B still caused disease in mice, but with delayed onset, whereas type A no longer caused disease. The rate of bacterial decline depended on subspecies, temperature, and the initial inoculum size. We further explored the persistence of type A and type B populations in natural pond water, which corroborated findings from axenic conditions and revealed that both strains perpetuated disease from a non-replicative inactive persistent state. This study provides timeframes for the persistence of virulent planktonic F. tularensis subspecies tularensis and holarctica populations in various aquatic conditions, contributing to increased understanding of host-independent survival as a component of the perpetuation of tularemia. IMPORTANCE Long-term persistence of planktonic (free living in water) Francisella tularensis subspecies tularensis (type A) and subspecies holarctica (type B) requires low temperatures and reduced microbial complexity. These conditions are typical of water wells, supporting the epidemiological observations that drinking water reservoirs may serve as sources of human F. tularensis infections (specifically type B). Importantly, persistence of types A and B did not correlate with molecular viability assays and culturability. This suggests that risk assessment of contagious bacteria based on these methods may not be appropriate. This is the first study to perform side-by-side comparisons of fully virulent strains of type A (Schu S4) and type B (FSC200), to evaluate persistence (defined by the capability to cause tularemia in mice) outside the host-restricted niche under stress conditions in water. The results contribute to the understanding of long-term persistence of F. tularensis between tularemia outbreaks in humans and other mammals.