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Background   Francisella (F.) tularensis  is the causative agent of tularemia. Due to its low infectious dose, ease of dissemination and high case fatality rate,  F. tularensis  was the subject in diverse biological weapons programs and is among the top six agents with high potential if misused in bioterrorism. Microbiological diagnosis is cumbersome and time-consuming. Methods for the direct detection of the pathogen (immunofluorescence, PCR) have been developed but are restricted to reference laboratories.    Results  The complete 23S rRNA genes of representative strains of  F. philomiragia  and all subspecies of  F. tularensis  were sequenced. Single nucleotide polymorphisms on species and subspecies level were confirmed by partial amplification and sequencing of 24 additional strains. Fluorescent In Situ Hybridization (FISH) assays were established using species- and subspecies-specific probes.  Different FISH protocols allowed the positive identification of all 4  F. philomiragia  strains, and more than 40  F. tularensis  strains tested. By combination of different probes, it was possible to differentiate the  F. tularensis  subspecies  holarctica, tularensis, mediasiatica  and  novicida . No cross reactivity with strains of 71 clinically relevant bacterial species was observed. FISH was also successfully applied to detect different  F. tularensis  strains in infected cells or tissue samples. In blood culture systems spiked with  F. tularensis , bacterial cells of different subspecies could be separated within single samples.    Conclusion  We could show that FISH targeting the 23S rRNA gene is a rapid and versatile method for the identification and differentiation of  F. tularensis  isolates from both laboratory cultures and clinical samples.

Rapid differentiation of Francisella species and subspecies by fluorescent in situ hybridization targeting the 23S rRNA