title: 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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
  
  
  BACKGROUND
  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.
  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).
  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.
authors: The Center for Microbial Genetics and Genomics, Northern Arizona University, Flagstaff, AZ 86011-4073, USA; The Pathogen Genomics Division, The Translational Genomics Research Institute, 3051 W. Shamrell Blvd, Suite 106, Flagstaff, AZ 86001 USA.
authors: Paul
authors: P
authors: Keim
authors: The Robert Koch Institute, Berlin, Germany.
authors: Roland
authors: R
authors: Grunow
authors: Public Health England, Porton Down, Wiltshire SP4 0JG, United Kingdom; NIHR Health Protection Research Unit in Emerging and Zoonotic Infections, Liverpool L69 7BE, United Kingdom.
authors: Richard
authors: R
authors: Vipond
authors: Bundeswehr Institute of Microbiology, Munich, Germany.
authors: Gregor
authors: G
authors: Grass
authors: The Center for Disease Control and Prevention, Atlanta, GA, USA.
authors: Alex
authors: A
authors: Hoffmaster
authors: The Center for Microbial Genetics and Genomics, Northern Arizona University, Flagstaff, AZ 86011-4073, USA.
authors: Dawn N
authors: DN
authors: Birdsell
authors: The Robert Koch Institute, Berlin, Germany.
authors: Silke R
authors: SR
authors: Klee
authors: Public Health England, Porton Down, Wiltshire SP4 0JG, United Kingdom; NIHR Health Protection Research Unit in Emerging and Zoonotic Infections, Liverpool L69 7BE, United Kingdom.
authors: Steven
authors: S
authors: Pullan
authors: Public Health England, Porton Down, Wiltshire SP4 0JG, United Kingdom.
authors: Markus
authors: M
authors: Antwerpen
authors: The Center for Microbial Genetics and Genomics, Northern Arizona University, Flagstaff, AZ 86011-4073, USA.
authors: Brittany N
authors: BN
authors: Bayer
authors: Public Health England, Porton Down, Wiltshire SP4 0JG, United Kingdom.
authors: Jennie
authors: J
authors: Latham
authors: The Pathogen Genomics Division, The Translational Genomics Research Institute, 3051 W. Shamrell Blvd, Suite 106, Flagstaff, AZ 86001 USA.
authors: Kristin
authors: K
authors: Wiggins
authors: The Center for Microbial Genetics and Genomics, Northern Arizona University, Flagstaff, AZ 86011-4073, USA.
authors: Crystal
authors: C
authors: Hepp
authors: The Center for Microbial Genetics and Genomics, Northern Arizona University, Flagstaff, AZ 86011-4073, USA.
authors: Talima
authors: T
authors: Pearson
authors: Public Health England, Porton Down, Wiltshire SP4 0JG, United Kingdom; NIHR Health Protection Research Unit in Emerging and Zoonotic Infections, Liverpool L69 7BE, United Kingdom.
authors: Tim
authors: T
authors: Brooks
authors: The Center for Microbial Genetics and Genomics, Northern Arizona University, Flagstaff, AZ 86011-4073, USA; The Pathogen Genomics Division, The Translational Genomics Research Institute, 3051 W. Shamrell Blvd, Suite 106, Flagstaff, AZ 86001 USA.
authors: Jason
authors: J
authors: Sahl
authors: The Center for Microbial Genetics and Genomics, Northern Arizona University, Flagstaff, AZ 86011-4073, USA.
authors: David M
authors: DM
authors: Wagner
mesh_terms: D000881:Anthrax / Q000453:epidemiology* / Q000382:microbiology* / Q000635:transmission; D001408:Bacillus anthracis / Q000145:classification* / Q000235:genetics*; D016000:Cluster Analysis; D004196:Disease Outbreaks; D055030:Drug Users; D016680:Genome, Bacterial; D023281:Genomics / Q000379:methods; D006801:Humans; D017720:Molecular Epidemiology; D010802:Phylogeny; D058974:Phylogeography; D020641:Polymorphism, Single Nucleotide; D017422:Sequence Analysis, DNA; D062211:Spatio-Temporal Analysis
keywords: Bacillus anthracis; Heroin; Injectional Anthrax; Phylogeny; SNP; Whole Genome Sequencing