Thus, rapid and reliable procedures for the

direct detection and differentiation of Francisellae in clinical samples may prove helpful to both clinicians and public health authorities. Therefore, a 23S rRNA-based detection approach was developed, since this molecule has been used extensively to elucidate phylogenetic relationships of bacteria at intra- and intergeneric levels and it is also an excellent target for fluorescent in situ hybridization [24–26]. Near full-length AZD0156 nmr 23S rRNA gene sequences for F. philomiragia and all four subspecies of F. tularensis were determined. Additional sequences for this target, which exists in three copies in the known Francisella genomes, were analyzed by extracting this information from the published whole genomes sequences currently available. These sequence data were used to develop additional primer sets and fluorescently labeled oligonucleotide probes suitable for species- and subspecies-specific fluorescent in situ hybridization (FISH) of pathogenic Francisella species in culture as well as clinical specimens. Methods Preparation

of samples for in situ hybridization and PCR All bacterial strains used in this study are listed in Table 1 and 2. Francisella strains were grown aerobically on heart cysteine LY2835219 in vitro agar (HCA) at 37°C and 5% CO2. All other strains were cultured on Columbia blood agar or in Luria-Bertani (LB) broth (BD, Heidelberg,

Germany). Bacterial cells were harvested while in exponential phase, suspended in phosphate buffered saline (PBS), centrifuged, washed in PBS, resuspended in TE buffer (10 mM Tris, 1 mM EDTA [pH8]), and adjusted to an optical density of 1.0 at 600 nm. Bacterial suspensions were prepared for PCR analysis using the QIAGEN (Hilden, Germany) tissue kit as recommended by the manufacturer. about For in situ hybridization, harvested cells were processed and fixed with paraformaldehyde (PFA) as previously described [27]. Table 1 Results of fluorescence in situ hybridization of all Francisella (F.) tularensis and F. philomiragia strains used in this study. Species and origin Strain Alt. designation Hybridization probe       Bwall 1448 Bwphi 1448 Bwhol 1151 Bwnov 168 Bwtume 168II Bwmed 1397 F. tul. subsp. tularensis                 Human, Ohio, 1941 FSC237 Schu S4 + – - – + – Squirrel, Georgia (USA) FSC033 SnMF + – - – + – Tick, BC, Canada, 1935 FSC041 Vavenby + – - – + – Canada FSC042 Utter + – - – + – Hare Nevada, 1953 FSC054 Nevada 14 + – - – + – Human, Utah, 1920 FSC230 ATCC 6223 + – - – + – F. tul. subsp.