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The allelic identities of Single Nucleotide Repeat (SNR) markers in Bacillus anthracis are typically ascertained by DNA sequencing through the direct repeat. Here we describe a reproducible method for genotyping closely related isolates by using four SNR loci in a multiplex-PCR capillary electrophoresis system amenable to high-throughput analysis.

Single nucleotide polymorphisms (SNPs) are increasingly recognized as important diagnostic markers for the detection and differentiation ofBacillus anthracis. The use ofSNP markers for identifying B. anthracis DNA in environmental samples containing genetically similar bacteria requires the ability to amplify and detect DNA with single nucleotide specificity. We designed a TaqMan® mismatch amplification mutation assay (TaqMAMA) around a SNP in the plcR gene ofB. anthracis. The assay permits specific, low-level detection (25fg DNA) of this B. anthracis-specific SNP, even in the presence of environmental DNA extracts containing a 20,000-fold excess of the alternate allele. We anticipate that the ability to selectively amplify and detect low copy number DNAs with single nucleotide specificity will represent a valuable tool in the arena ofbiodefense and microbialforensics.

Aims: Bacillus anthracis is a genetically monomorphic bacterium with little diversity to be expected during an outbreak. This study used more rapidly evolving genetic markers on outbreak samples to ascertain genetic diversity. Methods and Results: Forty-seven isolates from a B. anthracis outbreak during the summer of 2005 in South Dakota were analysed using single nucleotide polymorphisms (SNP) and multi-locus VNTR analysis (MLVA). Results indicated that all of the outbreak strains belonged to a single clonal lineage. However, analysis of four single nucleotide repeat (SNR) markers resolved these isolates into six distinct genotypes providing insights into disease transmission. Conclusions: Strain determination of unknown B. anthracis samples can be ascertained by SNP and MLVA markers. However, comparison of many samples obtained during an outbreak will require markers with higher rates of mutation to ascertain genetic diversity. Significance and Impact of the Study: SNR4 analysis allowed discrimination of closely related B. anthracis isolates and epidemiological tracking of the outbreak. When used in conjunction with other genotyping schemes that allow broad genetic relationships to be determined, SNR markers are powerful tools for detailed tracking of natural B. anthracis outbreaks and could also prove useful in forensic investigations.

Anthrax, caused by the bacterium Bacillus anthracis, is a disease of historical and current importance that is found throughout the world. The basis of its historical transmission is anecdotal and its true global population structure has remained largely cryptic. Seven diverse B. anthracis strains were whole-genome sequenced to identify rare single nucleotide polymorphisms (SNPs), followed by phylogenetic reconstruction of these characters onto an evolutionary model. This analysis identified SNPs that define the major clonal lineages within the species. These SNPs, in concert with 15 variable number tandem repeat (VNTR) markers, were used to subtype a collection of 1,033 B. anthracis isolates from 42 countries to create an extensive genotype data set. These analyses subdivided the isolates into three previously recognized major lineages (A, B, and C), with further subdivision into 12 clonal sub-lineages or sub-groups and, finally, 221 unique MLVA15 genotypes. This rare genomic variation was used to document the evolutionary progression of B. anthracis and to establish global patterns of diversity. Isolates in the A lineage are widely dispersed globally, whereas the B and C lineages occur on more restricted spatial scales. Molecular clock models based upon genome-wide synonymous substitutions indicate there was a massive radiation of the A lineage that occurred in the mid-Holocene (3,064-6,127 ybp). On more recent temporal scales, the global population structure of B. anthracis reflects colonial-era importation of specific genotypes from the Old World into the New World, as well as the repeated industrial importation of diverse genotypes into developed countries via spore-contaminated animal products. These findings indicate humans have played an important role in the evolution of anthrax by increasing the proliferation and dispersal of this now global disease. Finally, the value of global genotypic analysis for investigating bioterrorist-mediated outbreaks of anthrax is demonstrated.

We used multiple-locus variable-number tandem repeat analysis (MLVA) to type 64 Bacillus anthracis isolates from outbreaks that have occurred during the past 40 years in Italy. MLVA of the 64 isolates revealed 10 unique genotypes; 9 of these genotypes and the majority of isolates (63/64) belonged to the previously described genetic cluster A1.a. Within the A1.a isolates, two previously described genotypes (G1 and G3), which differ by a single mutation in the pX01 locus, account for the majority of isolates in the country (53/63). The low diversity of B. anthracis genotypes in Italy suggests a single, dominant historical introduction, followed by limited localized differentiation.

Background The global pattern of distribution of 1033 B. anthracis isolates has previously been defined by a set of 12 conserved canonical single nucleotide polymorphisms (canSNP). These studies reinforced the presence of three major lineages and 12 sub-lineages and sub-groups of this anthrax-causing pathogen. Isolates that form the A lineage (unlike the B and C lineages) have become widely dispersed throughout the world and form the basis for the geographical disposition of "modern" anthrax. An archival collection of 191 different B. anthracis isolates from China provides a glimpse into the possible role of Chinese trade and commerce in the spread of certain sub-lineages of this pathogen. Canonical single nucleotide polymorphism (canSNP) and multiple locus VNTR analysis (MLVA) typing has been used to examine this archival collection of isolates. Results The canSNP study indicates that there are 5 different sub-lineages/sub-groups in China out of 12 previously described world-wide canSNP genotypes. Three of these canSNP genotypes were only found in the western-most province of China, Xinjiang. These genotypes were A.Br.008/009, a sub-group that is spread across most of Europe and Asia; A.Br.Aust 94, a sub-lineage that is present in Europe and India, and A.Br.Vollum, a lineage that is also present in Europe. The remaining two canSNP genotypes are spread across the whole of China and belong to sub-group A.Br.001/002 and the A.Br.Ames sub-lineage, two closely related genotypes. MLVA typing adds resolution to the isolates in each canSNP genotype and diversity indices for the A.Br.008/009 and A.Br.001/002 sub-groups suggest that these represent older and established clades in China. Conclusion B. anthracis isolates were recovered from three canSNP sub-groups (A.Br.008/009, A.Br.Aust94, and A.Br.Vollum) in the western most portion of the large Chinese province of Xinjiang. The city of Kashi in this province appears to have served as a crossroads for not only trade but the movement of diseases such as anthrax along the ancient "silk road". Phylogenetic inference also suggests that the A.Br.Ames sub-lineage, first identified in the original Ames strain isolated from Jim Hogg County, TX, is descended from the A.Br.001/002 sub-group that has a major presence in most of China. These results suggest a genetic discontinuity between the younger Ames sub-lineage in Texas and the large Western North American sub-lineage spread across central Canada and the Dakotas.