Genome-Wide Single Nucleotide Polymorphism Typing Method for Identification of Bacillus anthracis Species and Strains among B. cereus Group Species

Abstract

As an issue of biosecurity, species-specific genetic markers have been well characterized. However, Bacillus anthracis strain-specific information is currently not sufficient for traceability to identify the origin of the strain. By using genome-wide screening using short read mapping, we identified strainspecific single nucleotide polymorphisms (SNPs) among B. anthracis strains including Japanese isolates, and we further developed a simplified 80-tag SNP typing method for the primary investigation of traceability. These 80-tag SNPs were selected from 2,965 SNPs on the chromosome and the pXO1 and pXO2 plasmids from a total of 19 B. anthracis strains, including the available genome sequences of 17 strains in the GenBank database and 2 Japanese isolates that were sequenced in this study. Phylogenetic analysis based on 80-tag SNP typing showed a higher resolution power to discriminate 12 Japanese isolates rather than the 25 loci identified by multiple-locus variable-number tandem-repeat analysis (MLVA). In addition, the 80-tag PCR testing enabled the discrimination of B. anthracis from other B. cereus group species, helping to identify whether a suspected sample originates from the intentional release of a bioterrorism agent or environmental contamination with a virulent agent. In conclusion, 80-tag SNP typing can be a rapid and sufficient test for the primary investigation of strain origin. Subsequent whole-genome sequencing will reveal apparent strain-specific genetic markers for traceability of strains following an anthrax outbreak.Many potential bioterrorism agents, including anthrax, present as pulmonary disease. Anthrax is caused by the spore-forming bacterium Bacillus anthracis, which is among the most severe zoonoses posing a serious threat to both public and animal health (7,14). B. anthracis belongs to the Bacillus cereus group of bacteria, which is composed of closely related Gram-positive organisms with highly divergent virulent properties (14,18). Infection with this bacterium can occur through the skin, gastrointestinal tract, or respiratory apparatus following contact, ingestion, or inhalation of spores, respectively (7, 14).As an issue of biosecurity, a comprehensive molecular diagnosis system is considered for detecting potential infectious agents. For most potential bioterrorism agents, species-specific genetic markers have been well characterized (9), but strain-specific information is not sufficient for traceability to identify the origin of the strain.A liquid suspension of B. anthracis was dispersed by the Aum Shinrikyo religious cult in Japan in 1993. The genotype of the B. anthracis isolate released was identical to that of the Sterne 34F2 strain, which is a member of the A3b diversity cluster (10). Fortunately, there were no victims of this attack because the strain was pXO2 plasmid defective and a low-virulent derivative used commercially in Japan to vaccinate animals against anthrax. The recent "postal anthrax attacks" in the United States aimed at the intentional release of B. anthracis ...

Population Analysis of Streptococcus suis Isolates from Slaughtered Swine by Use of Minimum Core Genome Sequence Typing

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Fourteen-Genome Comparison Identifies DNA Markers for Severe-Disease-Associated Strains of Clostridium difficile

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Application of High-Throughput Sequencing: Discovery of Informative SNPs to Subtype <i>Bacillus anthracis</i>

“…The advent of next-generation sequencing technologies and comparative genome analyses offers a powerful approach for identifying rare polymorphisms for the unbiased typing of highly clonal pathogens. Since 2007, and the identification by Van Ert et al of 13 "canonical" SNPs representing key phylogenetic positions along the B. anthracis phylogenetic SNP tree, a growing number of diagnostic SNPs has been discovered and used to survey B. anthracis diversity in nature [11] [24] [30]- [32]. In this study, a same but large-scale approach, comparing 161 B. anthracis genomes of diverse origins, were conducted to discover novel informative SNPs that can further discriminate within lineages or groups of strains.…”