Francisella tularensis does not manifest virulence in viable but non-culturable state

Abstract

Francisella tularensis is a small Gram‐negative bacterium that causes tularemia in animals and man. The disease can be transmitted by handling of infected animals, by contaminated dust, by insect vectors, or by drinking contaminated water. In the present study cells of F. tularensis were subjected to extended storage in cold water devoid of carbon sources. Total cell counts remained constant throughout a 70‐day period and beyond, while plate counts decreased to an undetectable level after 70 days. Attempts to resuscitate the cells were unsuccessful. Quantitative PCR targeting the 16S rDNA of F. tularensis showed an increase in variability after 25 days and the signal was lost after 45 days. Metabolic activity, measured by accumulation of rhodamine 123, declined to approximately 35% after a 140‐day period. Analyses of substrate responsiveness of cells stored for 140 days in cold water showed that approximately 30% of the population increased in size after incubation in rich medium in the presence of nalidixic acid. Approximately 105 of these cells were injected intraperitoneally into mice. No signs or symptoms of tularemia were observed during 3 weeks. In addition, there was no evidence of stimulation of lymphocytes with F. tularensis as recall antigen. In conclusion, viable but non‐culturable cells of F. tularensis are avirulent in mice, giving new insight into the ecological niche of this bacterium.

Survival and persistence of nonspore-forming biothreat agents in water

“…Following literature reports of improved survival at 8°C (Jellison et al. 1950; Forsman et al. 2000), the F. tularensis experiments thereafter were conducted only at 8°C.…”

“…Researchers conducting similar work with F. tularensis were successful with culture at 8°C and hypothesize that 8°C is closer than 5 or 25°C to temperatures found in mountain streams, a habitat in which the organism is commonly found (Jellison et al. 1950; Forsman et al. 2000).…”

“…2000). Forsman et al. 2000) found that F. tularensis LVS was culturable up to 70 days at 8°C in tap water, but the organism was found to be still metabolically active for up to 140 days, as determined by the accumulation of Rhodamine 123 and the response to nalidixic acid.…”

Quantum of infection of Francisella tularensis tularensis in host-seeking Dermacentor variabilis

“…Only live bacteria residing in the salivary glands would be immediately available for transmission via tick bite. The agent of tularemia also is known to enter a viable but non-cultivatable (VBNC) and presumably non-infectious state in the environment (Forsman et al, 2000), and it is possible that tick-bound F. tularensis is similarly in a VBNC state. On the other hand, homogenates of ticks containing F. tularensis DNA are capable of infecting mice and causing disease when injected; and, the agent may be recovered from such homogenates by cultivation on CHAB (Hopla, 1960).…”

Occurrence and Control of Tularemia in Drinking Water

“…F. tularensis subsp. tularensis is the more virulent subspecies and, with few exceptions (Johansson et al 2000, Guryčová 1998), it has been reported only from North America. F. tularensis subsp.…”