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

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.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.Tularemia is a zoonotic disease caused by the highly infectious, virulent, gram-negative bacterium F. tularensis. This bacterial disease occurs in various clinical forms depending on the route of inoculation and the virulence of the F. tularensis strain involved [1]. The geographical distribution of F. tularensis was long regarded to be restricted to the Northern Hemisphere [2], and only very recently F. tularensis-like strains have been cultured in Queensland, Australia [3], and Thailand, South-East Asia [4]. F. tularensis has a broad host range and can affect more animal species than any other zoonotic pathogen [2]. Whereas human infections in North America are mainly due to tick bites or contact with rabbits, several enzootic cycles have been described in the Eurasia. Here