In contrast to most other bacterial pathogens, cultivation of F. tularensis is difficult due to its fastidious nature and its susceptibility to overgrowth by concomitant flora. Additionally, growth may be delayed (up to 12 days) and cultivation of F. tularensis poses a significant threat of laboratory infections. EX 527 in vitro Only recently, conventional and real-time PCR protocols for the detection and identification of F. tularensis have been published, but still none of these techniques is sufficiently evaluated to be routinely used in clinical laboratories [36]. In this study we

evaluated the potential of rRNA gene targeted PCR and sequencing as well as fluorescent in situ hybridization for the detection and differentiation of Francisella species. In- silico analysis of partial and complete 16S rRNA genes available in publicly accessible databases like GenBank confirmed the results of a previous study by showing that 16S rRNA sequences from F. tularensis subspecies are almost identical, and therefore, are only of limited value for the detection and discrimination of F. tularensis on the species or subspecies level [32]. In this

NVP-BGJ398 clinical trial regard, the difficulties to discriminate type A and ACY-1215 order type B strains resembled the situation in the closely related zoonotic pathogens Yersinia (Y.) pseudotuberculosis all and Y. pestis or Burkholderia (B.) pseudomallei and B. mallei [25, 37, 38] In contrast to those studies, comparison of full-length 23S rRNA genes of all

F. tularensis subspecies as well as F. philomiragia revealed several discriminative SNPs. The sequence data obtained from rRNA gene sequences, known to be highly conserved in bacterial phylogeny, could be successfully used for the construction of hybridization probes, allowing a rapid genotype-based detection of Francisella species on different taxonomic levels. A unique 23S rRNA target region suitable for the detection of F. tularensis subsp. holarctica (type B) could be identified. For the discrimination of F. tularensis subsp. tularensis (type A) and subsp. mediasiatica, an identification approach was developed by employing two different probes. Six type A strains, 31 type B strains as well as three F. tularensis subsp. mediasiatica strains were correctly identified by this approach, whereas no false-positive signal was observed with 71 other variably related bacterial species. Similar results were gained employing species-specific probes for F. philomiragia and F. tularensis, which were tested with all mentioned F. tularensis strains as well as four F. philomiragia strains. We also developed an in situ hybridization protocol for F. tularensis subsp. novicida, which allowed the detection of all four available strains of this subspecies.