Intraspecific Differentiation of <I>Francisella tularensis</I> Strains Using Molecular-Genetic Methods. Complex Approach

Abstract

The aim of the study was to develop an algorithm for intraspecific differentiation of tularemia agent strains using a set of approaches based on amplification and sequencing technologies.Materials and methods. 97 strains of Francisella tularensis of various subspecies, biovars and subpopulations from the State Collection of Pathogenic Bacteria of the Russian Research Anti-Plague Institute “Microbe” were used in the work. The intraspecific identification of tularemia agent strains was carried out using the “F. tularensis-4c” system; analysis of the variability of the RD1 differentiation region, the sdhA gene, by applying the disk diffusion method using disks with erythromycin. Fragment Sanger sequencing was performed on a 3500 XL genetic analyzer (Applied Biosystems, USA) taking into account the manufacturer’s recommendations. Sequence homology assessment was conducted using the BLAST algorithm, the GenBank NCBI database, MEGA11 v11.0.13 and Unipro UGENE v50.0 software.Results and discussion. Subspecies- and biovarspecific mutations have been detected in the 23S rRNA gene. Promising regions of this gene for further investigation have been identified using fragment sequencing. A comprehensive scheme for intraspecific differentiation of tularemia microbe strains has been put forward, where at the first stage the subspecies and biovar japonica are determined, and at the second stage, the results are verified based on the determination of mutations in the 23S rRNA gene. The effectiveness of the proposed integrated approach has been confirmed in a study of 97 collection strains of tularemia agent. The conducted research allows for rapid identification of tularemia agent strains of different subspecies and verification of their taxonomic appurtenance using molecular-genetic methods, expanding data on the circulation of various subspecies, biovars and subpopulations of the pathogen in Europe, Asia and other regions of the world.

Keywords

• intraspecific differentiation
• francisella tularensis
• biovars
• subspecies
• algorithm
• integrated approach
• amplification and sequencing technologies