Abstract
The largest phylogenetic lineage known to date of the anthrax pathogen Bacillus anthracis is the wide-spread, so-called Trans-Eurasian clade systematically categorized as the A.Br.008/009 group sharing two defining canonical single-nucleotide polymorphisms (canSNP). In this study, we genome-sequenced a collection of 35 B. anthracis strains of this clade, derived from human infections, animal outbreaks or soil, mostly from European countries isolated between 1936 and 2008. The new data were subjected to comparative chromosomal analysis, together with 75 B. anthracis genomes available in public databases, and the relative placements of these isolates were determined within the global phylogeny of the A.Br.008/009 canSNP group. From this analysis, we have detected 3754 chromosomal SNPs, allowing the assignation of the new chromosomal sequences to established sub-clades, to define new sub-clades, such as two new Spanish, one Bulgarian or one German group(s), or to introduce orphan lineages. SNP-based results were compared with that of a multilocus variable number of tandem repeat analysis (MLVA). This analysis indicated that MLVA typing might provide additional information in cases when genomics yields identical genotypes or shows only minor differences. Introducing the delayed mismatch amplification assay (DMAA) PCR-analysis, we developed a cost-effective method to interrogate for a set of ten phylogenetically informative SNPs within genomes of A.Br.008/009 canSNP clade strains of B. anthracis. By this approach, additional 32 strains could be assigned to five of ten defined clades.
Highlights
Bacillus anthracis, the causative agent of the zoonotic infectious disease anthrax has a broad geographic distribution, mostly affecting regions in Africa, South America and Asia [1]
All live B. anthracis strains were handled in biosafety level 3 (BSL-3) laboratories at the Bundeswehr Institute of Microbiology (IMB) or University of Hohenheim
All data generated or analyzed during this study are included in this published article, and its supplementary information files are publically available in the NCBI Sequence Read Archive (SRA) repository (Bioproject PRJNA309927)
Summary
The causative agent of the zoonotic infectious disease anthrax has a broad geographic distribution, mostly affecting regions in Africa, South America and Asia [1]. Within the A-branch, the A.Br. 008/009 clade, i.e., strains phylogenetically defined by the two canonical (can) single nucleotide polymorphisms (SNPs) A.Br. and A.Br., is the one with the broadest geographical distribution This clade has been named the Trans-Eurasian branch [10] because it is dominant in most of Europe and (western) regions of Asia, even though we know today that some African [11] or American strains [8] are included. With the objective of generating an increasingly detailed picture of the phylogenetic population structure of the Trans-Eurasian A.Br.008/009 canSNP clade of B. anthracis, we have analyzed 35 strains from our strain collections on a genomic level This new information was used to describe the phylogenetic placement of these isolates within the A.Br.008/009 clade, and to design and test new SNP-based assays
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