see more tularensis subsp. tularensis Schu S4 and other AI strains. All type AII strains displayed the Francisella consensus sequence at this position. The phylogenetic tree generated using a neighbor joining analysis procedure (Fig. 2) is consistent with data from the 16S rRNA gene and a whole genome SNP phylogeny [5] showing that F. philomiragia is clearly distal from the F. tularensis subspecies. Among the latter,

strains of the subspecies F. tularensis subsp. novicida are distinct and derived from the ancestral lineage leading to the other subspecies. The phylogeny then separates into two lineages with F. tularensis subsp. tularensis and F. tularensis subsp. mediasiatica on one side and strains belonging to F. tularensis subsp. holarctica on the other. The type A strains can then be further separated into two groups corresponding to the known subtypes AI and AII. Figure 2 Phylogentic tree based on nearly complete Gamma-secretase inhibitor nucleotide Thiazovivin sequences

of 23S rRNA gene of F. tularensis and F. philomiragia generated using a neighbor joining analysis. Bootstrap values based on 1000 resamplings are indicated at branch points. Sensitivity and specificity of in situ hybridization Probe Bwall1448 was targeted to an rRNA region unique for the genus Francisella and gave a positive signal for all Francisella strains tested in this study (Table 1). Due to a single mismatch within the binding site of this probe in F. philomiragia strains, the simultaneous application of a second probe (Bwphi1448), labeled with a different fluorochrome

allowed the identification of all investigated Francisella strains on the species level by a single analysis (Fig. 3). Alternatively, each probe could be combined with EUB338, Reverse transcriptase a probe targeted to a 16S rRNA-sequence conserved in most bacterial species, to rapidly exclude the presence of either F. tularensis or F. philomiragia in a bacteria-containing sample. Figure 3 Left: Artificial mixture of F. tularensis subsp. tularensis (Schu S4, red circle) and F. philomiragia (ATCC 25017, green circle), phase contrast microscopy. Right: Fluorescence microscopy after hybridization with probes Bwall1448-Cy3 and Bwphi1448-6-FAM with 50% formamide. The green, pleomorphic cells of F. philomiragia can be easily distinguished from the smaller, coccoid rods of the highly virulent F. tularensis subsp. tularensis strain showing red fluorescence. All five available F. tularensis subsp. novicida strains hybridized to the probes Bwall1448 as well as the novicida-specific probe Bwnov168 that binds within helix 10b of the 23S rRNA. Probe Bwhol1151 (binding site nt 1151 to nt 1170, helix 45) is complementary to all F. tularensis holarctica sequences and gave strong signals when hybridized to its respective target strains (Table 1). No cross reactivity with F. tularensis subsp. tularensis or other subspecies were observed. The lack of suitable SNPs specific for all F. tularensis subsp.