Whole Genome Analysis of Injectional Anthrax Identifies Two Disease Clusters Spanning More Than 13 Years

Abstract

BackgroundAnthrax is a rare disease in humans but elicits great public fear because of its past use as an agent of bioterrorism. Injectional anthrax has been occurring sporadically for more than ten years in heroin consumers across multiple European countries and this outbreak has been difficult to trace back to a source.MethodsWe took a molecular epidemiological approach in understanding this disease outbreak, including whole genome sequencing of Bacillus anthracis isolates from the anthrax victims. We also screened two large strain repositories for closely related strains to provide context to the outbreak.FindingsAnalyzing 60 Bacillus anthracis isolates associated with injectional anthrax cases and closely related reference strains, we identified 1071 Single Nucleotide Polymorphisms (SNPs). The synapomorphic SNPs (350) were used to reconstruct phylogenetic relationships, infer likely epidemiological sources and explore the dynamics of evolving pathogen populations. Injectional anthrax genomes separated into two tight clusters: one group was exclusively associated with the 2009–10 outbreak and located primarily in Scotland, whereas the second comprised more recent (2012–13) cases but also a single Norwegian case from 2000.InterpretationGenome-based differentiation of injectional anthrax isolates argues for at least two separate disease events spanning > 12 years. The genomic similarity of the two clusters makes it likely that they are caused by separate contamination events originating from the same geographic region and perhaps the same site of drug manufacturing or processing. Pathogen diversity within single patients challenges assumptions concerning population dynamics of infecting B. anthracis and host defensive barriers for injectional anthrax.FundingThis work was supported by the United States Department of Homeland Security grant no. HSHQDC-10-C-00,139 and via a binational cooperative agreement between the United States Government and the Government of Germany. This work was supported by funds from the German Ministry of Defense (Sonderforschungsprojekt 25Z1-S-431,214). Support for sequencing was also obtained from Illumina, Inc. These sources had no role in the data generation or interpretation, and had not role in the manuscript preparation.Panel 1: Research in Context Systematic ReviewWe searched PubMed for any article published before Jun. 17, 2015, with the terms “Bacillus anthracis” and “heroin”, or “injectional anthrax”. Other than our previously published work (Price et al., 2012), we found no other relevant studies on elucidating the global phylogenetic relationships of B. anthracis strains associated with injectional anthrax caused by recreational heroin consumption of spore-contaminated drug. There were, however, publically available genome sequences of two strains involved (Price et al., 2012, Grunow et al., 2013) and the draft genome sequence of Bacillus anthracis UR-1, isolated from a German heroin user (Ruckert et al., 2012) with only limited information on the genotyping of closely relat...

Phylogenetic Placement of Isolates Within the Trans-Eurasian Clade A.Br.008/009 of Bacillus anthracis

“…Strain 319/02 separated 379 apomorphous SNPs from the A.Br.159 polytomy. Possibly, this isolate is a mutator strain, as described before for another strain with a similar enormous number of phylogenetically uninformative SNP-positions [27]. On the other hand, strain 319/02 was 373 SNPs and strain Sen3 359 SNPs, apart from their hypothetical last common ancestor (Table S2).…”

“…1,600,499; 1,979,090 and 5,013,862; Table S2; Figure 2c). Strain 3016, from Iran, is not particularly closely related to the strains associated with injectional anthrax outbreaks in Europe from 2000 until 2013 [27], as the strain 3016 chromosome was separated by 30 to 41 SNPs from outbreak strains (Figure 2c). Instead, strain 3016 shared a common hypothetical ancestor with strains A0897 (Ethiopia; 36 SNPs distance) and PAK-1 (Pakistan; 32 SNPs distance), respectively (Figures 1 and 2c).…”

Insights from Bacillus anthracis strains isolated from permafrost in the tundra zone of Russia

“…The first is defined by the Yakut 4Ya strain, the second is present mostly in China, and the third corresponds to the STI vaccine strain from Russia. The Tsiankowskii vaccine strain, the Sverdlosk 1979 strain [34], and the Yakutia 3Ya strain which is closest to a strain from Norway belong to lineage L3_Tsiankowskii lineage containing a single deep-branching lineage widely spreading in Russia and Eastern Europe, including Greece, Albania, Bulgaria, Poland. Lineage L4_Pasteur contains two deep-branching sublineages, one found in Bulgaria and the other in Italy in addition to the Pasteur I vaccine strain.…”

Injectional anthrax in human: A new face of the old disease

“…But the 10-year lapse in G-II injectional anthrax cases suggests that there is a single contaminating source for the G-II cases but that there have been 2 or more contamination events. 29…”

“…At least 3 possibilities are mentioned: via addition of cutting agent derived from an animal, such as bone meal, via wrapping in animal hide contaminated with anthrax spores, or via contamination with soil during drug manufacturing and trafficking. 25,29,30 At the beginning of the anthrax epidemic in Europe, it was suspected that the heroin contaminated with anthrax spores came from Afghanistan, where it was contaminated at the primary source, for example during raw heroin production, as Afghanistan produces 90% of the world's heroin. 29 However, the isolates from heroin users were excluded by genotyping from the Vollum branches strains commonly found in Afghanistan and Pakistan.…”