Development of a method for molecular subtyping <i>Bacillus anthracis</i> using HRM PCR

Introduction. Bacillus anthracis is the causative agent of anthrax, a pathogen characterized by high genetic monomorphism that complicates differentiation of strains. Thus, molecular methods for pathogen typing require the improvement. The aim of the study. To select marker SNPs for new genetic groups of B. anthracis and to develop a method for their laboratory identification using HRM PCR. Materials and methods. The core genome of 222 strains of B. anthracis from the GenBank database and 66 strains from the collection of pathogenic microorganisms of the Stavropol Anti-Plague Institute was aligned using the parsnp software. A dendrogram based on 7242 core genome SNPs was built in MEGA X software. The strains for validation of the HRM method included representatives of various genetic groups. The HRM PCR reaction was performed using the "Type-it HRM PCR Kit" and "KAPA HRM FAST qPCR Kit" and a Rotor Gene DNA thermocycler with HRM function. Data analysis and visualization were performed using custom scripts in the Python and R development environments. Results and discussion. Marker SNPs for 6 genetic groups have been identified, which make it possible to determine whether strains belong to one of 7 new subclusters. Pairs of primers were selected for the loci containing them, HRM PCR parameters were optimized for discrimination of different alleles of SNP loci, and an analysis scheme was developed. Conclusion. Thus, marker SNPs were selected to determine the genetic subclusters A.Br.CEA, A.Br.STI, A.Br.Tsiankovskii, B.Br.Europe, B.Br.Siberia, B.Br.Asia, B.Br.018, and a new laboratory method was developed for molecular subtyping of B. anthracis using HRM PCR

<i>In silico</i> analysis of genomes of Bacillus anthracis strains belonging to major genetic lineages

Introduction. The global phylogenetic population structure of Bacillus anthracis is represented by major genetic lineages (A, B and C) with nonuniform distribution of isolates, which still cannot be explained. Identification of characteristics of genomes of strains from three lineages, which can affect their spread, is of high importance. The aim of the study is to explore genomic characteristics of different genetic lineages, which may have an effect on their distribution, by using the in silico analysis of a representative subset of B. anthracis strains. Materials and methods. The whole-genome sequences of 49 B. anthracis strains and Bacillus cereus biovar anthracis CI strain were studied. The in silico analysis was performed to identify polymorphisms using BLASTn, MEGA X, Tandem Repeat Finder, Parsnp the Harvest Suite software. Results. The genome variability depended on single nucleotide polymorphisms, single-nucleotide repeats, number of tandem repeats, substitutions and indels. In strains from lineages B and C, they outnumbered 1.613.4 times and in the B. cereus biovar anthracis strain 5150 times those in B. anthracis strains from lineage A. Significant substitutions in housekeeping genes and pathogenicity factor genes caused changes in amino acid sequences in proteins significantly more frequently in B. anthracis strains from major lineages B and C. Based on the molecular typing and a multi-virulence-locus sequence typing analysis (MVLST) with a discrimination index of 0.9633, strains were classified into three major genetic lineages including groups different from the canonical group. Conclusion. The distinctive feature of B. anthracis genomes is that they have a larger number of significant nucleotide substitutions in pathogenicity factor genes and housekeeping genes of strains belonging to major lineages B and C compared to lineage A. Changes in proteins encoded by them can cause differences in ecological adaptation and in prevalence, which are higher in strains of lineage A. MVLST having a high discriminating capacity can be used as an additional method to B. anthracis molecular typing