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

Francisella tularensis is a small Gram-negative bacterium that causes tular emia in animals and man. The disease can be transmitted by handling of infe cted animals, by contaminated dust, by insect vectors, or by drinking conta minated water. In the present study cells of F. tularensis were subjected t o extended storage in cold water devoid of carbon sources. Total cell count s remained constant throughout a 70-day period and beyond, while plate coun ts decreased to an undetectable level after 70 days. Attempts to resuscitat e 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 rh odamine 123, declined to approximately 35% after a 140-day period. Analyses of substrate responsiveness of cells stored for 140 days in cold water sho wed that approximately 30% of the population increased in size after incuba tion in rich medium in the presence of nalidixic acid. Approximately 10(5) of these cells were injected intraperitoneally into mice. No signs or sympt oms of tularemia were observed during 3 weeks. In addition, there was no ev idence of stimulation of lymphocytes with F. tularensis as recall antigen. In conclusion, viable but non-culturable cells of F. tularensis are avirule nt in mice, giving new insight into the ecological niche of this bacterium. (C) 2000 Federation of European Microbiological Societies. Published by El sevier Science B.V. All rights reserved.