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A robust, rapid and flexible real-time PCR assay for hierarchical genetic typing of clinical and environmental isolates of Francisella is presented. Typing markers were found by multiple genome and gene comparisons, from which 23 canonical single nucleotide polymorphisms (canSNPs) and 11 canonical insertion-deletion mutations (canINDELs) were selected to provide phylogenetic guidelines for classification from genus to isolate level. The specificity of the developed assay, which uses 68 wells of a 96-well real-time PCR format with a detection limit of 100 pg DNA, was assessed using 62 Francisella isolates of diverse genetic and geographical origins. It was then successfully used for typing 14 F. tularensis subsp. holarctica isolates obtained from tularemia patients in Sweden in 2008 and five more genetically diverse Francisella isolates of global origins. When applied to human ulcer specimens for direct pathogen detection the results were incomplete due to scarcity of DNA, but sufficient markers were identified to detect fine-resolution differences among F. tularensis subsp. holarctica isolates causing infection in the patients. In contrast to other real-time PCR assays for Francisella, which are typically designed for specific detection of a species, subspecies, or strain, this type of assay can be easily tailored to provide appropriate phylogenetic and/or geographical resolution to meet the objectives of the analysis. [ABSTRACT FROM AUTHOR]

Francisella tularensis is a potent mammalian pathogen well adapted to intracellular habitats, whereas F. novicida and F. philomiragia are less virulent in mammals and appear to have less specialized lifecycles. We explored adaptations within the genus that may be linked to increased host association, as follows. First, we determined the genome sequence of F. tularensis subsp. mediasiatica, the only subspecies that had not been previously sequenced. This genome, and those of 12 other F. tularensis isolates, were then compared to the genomes of F. novicida (three isolates) and F. philomiragia (one isolate). Signs of homologous recombination were found in ~19.2% of F. novicida and F. philomiragia genes, but none among F. tularensis genomes. In addition, random insertions of insertion sequence elements appear to have provided raw materials for secondary adaptive mutations in F. tularensis, e.g. for duplication of the Francisella Pathogenicity Island and multiplication of a putative glycosyl transferase gene. Further, the five major genetic branches of F. tularensis seem to have converged along independent routes towards a common gene set via independent losses of gene functions. Our observations suggest that despite an average nucleotide identity of >97%, F. tularensis and F. novicida have evolved as two distinct population lineages, the former characterized by clonal structure with weak purifying selection, the latter by more frequent recombination and strong purifying selection. F. tularensis and F. novicida could be considered the same bacterial species, given their high similarity, but based on the evolutionary analyses described in this work we propose retaining separate species names. [ABSTRACT FROM AUTHOR]

The F. tularensis type A strain FSC198 from Slovakia and a second strain FSC043, which has attenuated virulence, are both considered to be derivatives of the North American F. tularensis type A strain SCHU S4. These strains have been propagated under different conditions: the FSC198 has undergone natural propagation in the environment, while the strain FSC043 has been cultivated on artificial media in laboratories. Here, we have compared the genome sequences of FSC198, FSC043, and SCHU S4 to explore the possibility that the contrasting propagation conditions may have resulted in different mutational patterns. We found four insertion/deletion events (INDELs) in the strain FSC043, as compared to the SCHU S4, while no single nucleotide polymorphisms (SNPs) or variable number of tandem repeats (VNTRs) were identified. This result contrasts with previously reported findings for the strain FSC198, where eight SNPs and three VNTR differences, but no INDELs exist as compared to the SCHU S4 strain. The mutations detected in the laboratory and naturally propagated type A strains, respectively, demonstrate distinct patterns supporting that analysis of mutational spectra might be a useful tool to reveal differences in past growth conditions. Such information may be useful to identify leads in a microbial forensic investigation.