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

Abstract

Background: Anthrax 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.

Methods: We 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.

Findings: Analyzing 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.

Interpretation: Genome-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.

Funding: This 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 review: We 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 related strains (Price et al., 2012, Grunow et al., 2013).

Lay person interpretation: Injectional anthrax has been plaguing heroin drug users across Europe for more than 10 years. In order to better understand this outbreak, we assessed genomic relationships of all available injectional anthrax strains from four countries spanning a > 12 year period. Very few differences were identified using genome-based analysis, but these differentiated the isolates into two distinct clusters. This strongly supports a hypothesis of at least two separate anthrax spore contamination events perhaps during the drug production processes. Identification of two events would not have been possible from standard epidemiological analysis. These comprehensive data will be invaluable for classifying future injectional anthrax isolates and for future geographic attribution.

Keywords: Bacillus anthracis; Heroin; Injectional Anthrax; Phylogeny; SNP; Whole Genome Sequencing.

Fig. 1

Whole Genome Phylogenetic Reconstruction of Injectional Anthrax (IA) B. anthracis Isolates. SNPs discovered from whole genome sequences of 60 B. anthracis isolates (isolation dates in parentheses) sampled from 36 IA patients, seven closely related B. anthracis isolates, and one outgroup genome were used to reconstruct phylogenetic relationships (68 OTUs). Of the 1079 SNPs, 350 synapomorphic SNPs were phylogenetically informative for determining the tree topology. Sixteen SNPs separated the two IA groups. There was only a single homoplastic SNP in this phylogenetic model (CI = 0.997), which did not affect the topology. The Injectional Anthrax Subclade is labeled and consistent with the Price et al. (Price et al., 2012) definition, which was based upon two branch-specific SNPs (SNP1173928 and SNP1053700) marked here with an asterisk (*). Variation within IA-Group I and IA-Group II is presented in Fig 2 and Fig S1.

Fig. 2

Phylogenetic Diversity Within the Two Injectional Anthrax Groups. The diversity within each of the two IA groups is represented as dendrograms where the predicted ancestral genomes are the left vertical lines. Genomes perfectly matching the ancestral allele states are flush with these lines, whereas genomes differing by one or more SNPs have a horizontal line of the appropriate length. Group I (N = 49) contains 27 strains with genomes identical to the ancestor and 22 strains with genomes that differ from the ancestor, including 16 unique derived types. Shared SNPs (synapomorphic) identify 6 derived pairs. For Group I, independent isolates from the same patient (replicate genomes) are labeled into four different categories of within single patient diversity. Group II (N = 11) has six ancestral genome types and five genomes with derived allele states. The single B. anthracis clinical tissue specimen (P35 A4569) is labeled with an asterisk (*) and was not WGS but rather analyzed phylogenetically by the 11 new canSNP assays (Table S3) and, thus, is collapsed into the ancestral genome. There is only one incidence of allelic variation within patient replicate isolates for Group II.

Fig. 3

Distribution of Heroin-Associated Anthrax Cases in this Study. The location of the 37 IA cases (from which a total of 61 patient isolates were retrieved) is indicated along with their membership in Group I or II.