Genome-scale comparison of Francisella tularensis strains isolated in an endemic region of Spain

Abstract

[Background and aim]: Tularemia is caused by the gram-negative intracellular pathogen Francisella tularensis. In Europe, the region of Castilla y León, Northwest Spain, is a major hotspot for tularemia, where the largest outbreaks of the disease have been recently reported. While rodents and lagomorphs are recognised as the main mammalian hosts in Europe, the common voles (Microtus arvalis) are documented key agents for human tularemia in Northwestern Spain, as evidenced by a spatial and temporal coincidence between human cases and increases in vole abundance. This study aimed to perform comparative genomics of F. tularensis isolates from tissue samples of F. tularensis positive voles with 11 human isolates from 2014, when an increased number of human cases was observed in the same area of Spain. [Material and Methods]: For this study, we selected thirty-four trapped voles, sampled in 80 km2 of farmland in Palencia Province, Spain (42°1′N, 4°42′W), that tested Ft-positive, by conventional PCR and hybridization by reverse line blotting (targeting lpnA), and the multi-target TaqMan PCR, tul4 and ISFtu2 assays. Tissues (liver, spleen and lung) from each animal were minced, inoculated in chocolate agar PolyViteXTM at 37°C in 5% CO2, and observed at 24, 48 and 72h post-inoculation. So far, after DNA extraction, four isolates, showing typical Ft growth, were subjected to paired-end whole-genome sequencing in an Illumina MiSeq apparatus, followed by genome assembly and bioinformatics analysis. [Results and conclusions]: All trapped voles tested positive for F. tularensis subsp. holarctica. A preliminary core-genome SNPs-based analysis, representing >99% of the genome, showed that all isolates (N=15) were distinguishable by 33 single nucleotide variant sites, representing two clear phylogenetic clusters. Noteworthy, four newly sequenced vole-isolated strains segregated together with five human-associated strains from 2014, suggesting the potential persistence of this strain in this region.