Development of a technique for molecular typing of Bacillus anthracis strains using new VNTR and INDEL markers

Abstract

Introduction. Bacillus anthracis, the pathogen of a particularly dangerous zoonotic disease known as anthrax, requires strict epidemiological control and is characterized by high genetic homogeneity, which necessitates the development of genotyping methods. The aim of the study were to to find and characterize the VNTR and INDEL loci of B. anthracis and to develop on their basis a genotyping technique by PCR with electrophoretic detection of the results. Materials and methods. Marker search and phylogenetic analysis were performed on a sample of 388 genomes of B. anthracis strains, 322 from the GenBank collection (RefSeq) and 66 from the collection of the Stavropol Anti-Plague Institute of Rospotrebnadzor. Phylogenetic analysis was performed on the basis of SNP crustal alignment using the Parsnp program. The search for markers was carried out using the Mauve program and author's scripts in Python. PCR was performed using a ScreenMix-HS kit (CJSC "Eurogen", Russia). Results. Genomic variations of B. anthracis strains (SNP — 25,664, SNR — 14,387, VNTR — 693, INDEL — 14,667) were found, bioinformatic analysis of which revealed nine new VNTR and six INDEL molecular markers most suitable for genotyping. The genetic (allelic) variants of the markers are described. Primers were selected for the found markers and a PCR protocol with detection by electrophoresis in agarose gel was developed. When typing using VNTR markers was applied, the strains were divided into nine clusters: A.Br.Ames, A.Br.001/002, A.Br.Aust94, A.Br.005/006, A.Br.008/009 (Tsiankovskii), A.Br.008/009 (STI), A.Br.008/009 (A.Br.125), A.Br.008/009 (strain 228/269), B.Br.001/002. When typing using INDEL markers, the strains were divided into six clusters: A.Br.Ames, A.Br.001/002, A.Br.Aust94, A.Br.008/009(Tsiankovskii), B.Br.001/002(B.Br.014), as well as a cluster comprising several genetic lineages: A.Br.008/009 (STI), A.Br.008/009 (A.Br.125), A.Br.005/006 и B.Br.001/002. Conclusion. The use of the developed methodology for the identification of variable VNTR and INDEL loci makes it possible to reliably determine the phylogenetic position of B. anthracis strains and is promising for use in the epidemiological investigation of anthrax outbreaks.