Optimization of the Multilocus Sequence Typing Method for Virulence Genes of Bacillus anthracis (MVLST)

In Russia, the epidemic situation on anthrax remains unstable despite achieved progress in its prevention and treatment. Regardless of the nature of the outbreak, epidemiological investigation requires establishing the probable origin and genetic relatedness of the strain responsible, which is attained through molecular typing. Bacillus anthracis is characterized by high genetic monomorphism, complicating molecular typing. The MLST variant, a multilocus sequence typing method for virulence genes (MVLST), allows for effective differentiation of pathogen strains. The aim of this study was to select the optimal MVLST scheme for the genetic typing of B. anthracis. Materials and methods. The study used complete genomes of 49 B. anthracis strains. Phylogenetic trees were constructed using UPGMA algorithm through identifying SNPs with PhyloViz software. Protein virulence assessment and gene selection were performed using the online resource “VirulentPred Prediction of Prokaryotic Virulent Proteins”. The Hunter-Gaston discriminatory index (D) was calculated online using the Discriminatory Power Calculator program. Results and discussion. The MVLST-pXO1 scheme included 5 genes: lef, cya, pagA, atxA, gerXC, located on the pXO1 toxin plasmid. Typing with the MVLST-pXO1 scheme divided 49 strains into 21 genotypes (D=0.9209). The MVLST-pXO2 scheme covered 6 genes: 4 structural genes of capsule polypeptide (capA, capB, capC, capD), and 2 regulatory genes (acpA, acpB) on the pXO2 capsule plasmid. Typing with this scheme distinguished 14 genotypes (D=0.6675) among 49 strains. The derived MVLST-15 scheme provided higher discriminatory power compared to MVLST-19, dividing 49 B. anthracis strains into 40 genotypes (D=0.9864), whereas MVLST-19 identified 33 genotypes (D=0.9633). These results demonstrate that the MVLST-15 scheme is optimal, as it provides better discrimination of B. anthracis strains using a smaller set of loci while maintaining the classical phylogenetic structure of the anthrax pathogen.

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