Occurrence of Genetic Determinants Associated with Attenuation in Wild-Type Strains of the Tularemia Microbe

The aim of our work was to determine genetic markers associated with attenuation (deletions in RD-18, RD-19 positions, single mutations) in natural strains of the causative agent of tularemia. Materials and methods . The work used 107 Francisella tularensis strains from the State Collection of Pathogenic Bacteria of the Russian Anti-Plague Institute “Microbe”, as well as 1077 F. tularensis genomes provided by the NCBI GenBank database. Cultivation was carried out on Petri dishes with FT-agar (State Research Center of Applied Microbiology and Biotechnology), pH 7.2; the cultures were incubated for 36–48 h at a temperature of (37±1) °С. The draft genome was assembled using the Unicycler v.0.4.7 software package. The snippy v.4.6.0 program was used to obtain a matrix of core SNPs. The dendrogram was drawn in the PhyML program using the Likelihood method with the GTR substitution model. Results and discussion . Among the studied strains of tularemia microbe, eleven erythromycin-resistant strains of F. tularensis of the phylogenetic groups B.12 B.24 and B.12 B.20 were identified, containing deletions in the RD-18 and RD-19 regions. Erythromycinresistant and erythromycin-sensitive cultures of the pathogen of the groups B.12 B.24, B.12 B.20, B.12 B.39 and B.4, B.6 B.10, B.6 B.7, respectively, were also identified, having a deletion only in the RD-18 region – 11 strains, and only in RD-19 – 8. Of the 11 cultures with deletions in both regions, in 9 cases the strains belonged to the phylogenetic group B.12 B.24 and formed a separate cluster, the remaining two – to B.12 B.20. The cluster of such strains is characterized by the presence of seven unique mutations associated with the formation of lipopolysaccharide, metabolism of aromatic compounds and reproduction in macrophages. Three additional specific mutations have been identified for the B.12 B.24 group strains. We have identified 29 strains of the tularemia pathogen (vaccine ones, their derivatives and natural ones), carrying genetic markers associated with attenuation to varying degrees.