Differentiation of the Francsella tularensis subspecies by the INDEL typing method

Abstract

Background. Francisella tularensis, the etiological agent of tularemia, belongs to the facultative intracellular pathogens that cause severe disease in humans and man species of animals, and is a category A bioterrorism agent. Currently, F. tularensis is divided into four subspecies: F. tularensis subsp. tularensis (nearctica), F. tularensis subsp. holarctica, F. tularensis subsp. mediasiatica, F. tularensis subsp novicida, which differ in their pathogenicity and geographical distribution. Historically, this division was due to the different distribution area of strains, their differences in biochemical activity and pathogenicity for different hosts. The biochemical identification of subspecies is very laborious and requires work with live cultures of the microorganism, which determines the need to develop new molecular genetic approaches for genotyping F. tularensis strains.The aim of this study is to develop a method for differentiating subspecies and individual groups of F. tularensis based on INDEL typing. Research objectives: creation of a local database of nucleotide sequences of F. tularensis strains of different subspecies, search for INDEL markers that are significant for the differentiation of subspecies of the causative agent of tularemia, designing primers for the detection of INDEL markers using PCR, optimization of the set of INDEL markers and elucidation of phylogenetic relationships between the studied strains based on the proposed INDEL typing method.Materials and methods. The local database of nucleotide sequences of F. tularensis strains of different subspecies for comparative analysis of F. tularensis genomes presented in the GenBank database was created using the author's software. Detection of INDEL markers in the genomes of strains of the local database was carried out using the GeneExpert program. Primer design and in silico PCR were performed using the Primer3Plus software and the proprietary VirtualPCR software. Cluster analysis and construction of a phylogenetic tree were performed using the GrapeTree program.Results and discussion. The implementation of the proposed five INDEL markers for genotyping of 29 studied strains of different subspecies from the GenBank database made it possible to detect 9 individual genotypes with a high diversity index (DI = 0.85). Not only the corresponding division of the tularensis, holarctica, mediasiatica, and novicida subspecies into different clusters was noted, but also the intraspecific division into groups of strains was observed. Differentiation of F. tularensis subspecies was confirmed in vitro for the collection of strains of different subspecies of the Collection of Living Cultures of the Rostov-on-Don Plague Control Researsh Institute.Conclusion. For the first time, the F. tularensis subspecies differentiation system based on the INDEL typing method has been developed, which allows in vitro identification of both F. tularensis subspecies (tularensis, holarctica, mediasiatica and novicida) and groups of strains within subspecies without the need for strain sequencing. The method is protected by a patent. The topology of the INDEL phylogenetic tree of genotypes of F. tularensis strains correlates with the patterns of evolution of the tularemia microbe presented earlier. The proposed method can be used for combined typing of F. tularensis strains together with MLVA or SNP typing