Skip to main content

Advertisement

Log in

Source tracking of an anthrax outbreak in northeastern China using complete genome analysis and MLVA genotyping

  • Article
  • Published:
European Journal of Clinical Microbiology & Infectious Diseases Aims and scope Submit manuscript

Abstract

Anthrax is caused by Bacillus anthracis, an etiological agent behind zoonotic diseases worldwide. B. anthracis is also one of the most dangerous bioterrorism agents. An anthrax outbreak took place in Liaoning Province in northeastern China in August 2012. It resulted in seven human infections and dozens of dead cows. One B. anthracis strain, named Han, was isolated from a dead cow. This strain showed minor pathogenicity in mice and was suspected to be derived from the locally administered vaccine strain, Vac. In order to determine if the Han isolate was derived from the vaccine strain Vac and to track the source of the anthrax outbreak and, so, exclude the possibility of terrorism attack, a complete genome sequencing of these two B. anthracis strains was conducted. With the genome sequencing data, canonical single nucleotide polymorphism (SNP) analysis and whole-genome SNP screening were performed. The results indicate that the Han strain was markedly different from the Vac strain. Further analysis by multiple-locus variable-number tandem repeat analysis (MLVA) showed that Han clustered with previously reported Chinese strains. The result of MLVA15 confirmed that the Han strain is a naturally occurring isolate instead of an engineered agent deliberately distributed by terrorists or other parties. In conclusion, our method used in this study not only facilitates epidemiological studies but also made it easier to distinguish naturally occurring outbreaks from intentionally released pathogens.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  1. Okutani A, Sekizuka T, Boldbaatar B, Yamada A, Kuroda M, Inoue S (2010) Phylogenetic typing of Bacillus anthracis isolated in Japan by multiple locus variable-number tandem repeats and the comprehensive single nucleotide polymorphism. J Vet Med Sci 72(1):93–97

    Article  CAS  PubMed  Google Scholar 

  2. Ciammaruconi A, Grassi S, De Santis R, Faggioni G, Pittiglio V, D’Amelio R, Carattoli A, Cassone A, Vergnaud G, Lista F (2008) Fieldable genotyping of Bacillus anthracis and Yersinia pestis based on 25-loci Multi Locus VNTR Analysis. BMC Microbiol 8(1):21

    Article  PubMed  PubMed Central  Google Scholar 

  3. Lista F, Faggioni G, Valjevac S, Ciammaruconi A, Vaissaire J, Le Doujet C, Gorgé O, De Santis R, Carattoli A, Ciervo A (2006) Genotyping of Bacillus anthracis strains based on automated capillary 25-loci multiple locus variable-number tandem repeats analysis. BMC Microbiol 6(1):33

    Article  PubMed  PubMed Central  Google Scholar 

  4. Inglesby TV, O’Toole T, Henderson DA, Bartlett JG, Ascher MS, Eitzen E, Friedlander AM, Gerberding J, Hauer J, Hughes J, McDade J, Osterholm MT, Parker G, Perl TM, Russell PK, Tonat K; Working Group on Civilian Biodefense (2002) Anthrax as a biological weapon, 2002: updated recommendations for management. JAMA 287(17):2236–2252

    Article  PubMed  Google Scholar 

  5. Mock M, Fouet A (2001) Anthrax. Annu Rev Microbiol 55(1):647–671

    Article  CAS  PubMed  Google Scholar 

  6. Read TD, Peterson SN, Tourasse N, Baillie LW, Paulsen IT, Nelson KE, Tettelin H, Fouts DE, Eisen JA, Gill SR, Holtzapple EK, Okstad OA, Helgason E, Rilstone J, Wu M, Kolonay JF, Beanan MJ, Dodson RJ, Brinkac LM, Gwinn M, DeBoy RT, Madpu R, Daugherty SC, Durkin AS, Haft DH, Nelson WC, Peterson JD, Pop M, Khouri HM, Radune D, Benton JL, Mahamoud Y, Jiang L, Hance IR, Weidman JF, Berry KJ, Plaut RD, Wolf AM, Watkins KL, Nierman WC, Hazen A, Cline R, Redmond C, Thwaite JE, White O, Salzberg SL, Thomason B, Friedlander AM, Koehler TM, Hanna PC, Kolstø AB, Fraser CM (2003) The genome sequence of Bacillus anthracis Ames and comparison to closely related bacteria. Nature 423(6935):81–86

    Article  CAS  PubMed  Google Scholar 

  7. Turnbull PC (1999) Definitive identification of Bacillus anthracis—a review. J Appl Microbiol 87(2):237–240

    Article  CAS  PubMed  Google Scholar 

  8. Kuroda M, Serizawa M, Okutani A, Sekizuka T, Banno S, Inoue S (2010) Genome-wide single nucleotide polymorphism typing method for identification of Bacillus anthracis species and strains among B. cereus group species. J Clin Microbiol 48(8):2821–2829

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Rasko DA, Altherr MR, Han CS, Ravel J (2005) Genomics of the Bacillus cereus group of organisms. FEMS Microbiol Rev 29(2):303–329

    CAS  PubMed  Google Scholar 

  10. Van Ert MN, Easterday WR, Huynh LY, Okinaka RT, Hugh-Jones ME, Ravel J, Zanecki SR, Pearson T, Simonson TS, U’Ren JM, Kachur SM, Leadem-Dougherty RR, Rhoton SD, Zinser G, Farlow J, Coker PR, Smith KL, Wang B, Kenefic LJ, Fraser-Liggett CM, Wagner DM, Keim P (2007) Global genetic population structure of Bacillus anthracis. PLoS One 2(5):e461

    Article  PubMed  PubMed Central  Google Scholar 

  11. Keim P, Price LB, Klevytska AM, Smith KL, Schupp JM, Okinaka R, Jackson PJ, Hugh-Jones ME (2000) Multiple-locus variable-number tandem repeat analysis reveals genetic relationships within Bacillus anthracis. J Bacteriol 182(10):2928–2936

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Eitzen EM (1917) Use of biological weapons. Med Asp Chem Biol Warf 41(2):450

    Google Scholar 

  13. Pile JC, Malone JD, Eitzen EM, Friedlander AM (1998) Anthrax as a potential biological warfare agent. Arch Intern Med 158(5):429–434

    Article  CAS  PubMed  Google Scholar 

  14. Hoffmaster AR, Fitzgerald CC, Ribot E, Mayer LW, Popovic T (2002) Molecular subtyping of Bacillus anthracis and the 2001 bioterrorism-associated anthrax outbreak, United States. Emerg Infect Dis 8(10):1111–1116

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Bush LM, Abrams BH, Beall A, Johnson CC (2001) Index case of fatal inhalational anthrax due to bioterrorism in the United States. N Engl J Med 345(22):1607–1610

    Article  CAS  PubMed  Google Scholar 

  16. Keim P, Van Ert MN, Pearson T, Vogler AJ, Huynh LY, Wagner DM (2004) Anthrax molecular epidemiology and forensics: using the appropriate marker for different evolutionary scales. Infect Genet Evol 4(3):205–213

    Article  CAS  PubMed  Google Scholar 

  17. Beecher DJ (2006) Forensic application of microbiological culture analysis to identify mail intentionally contaminated with Bacillus anthracis spores. Appl Environ Microbiol 72(8):5304–5310

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Okinaka RT, Henrie M, Hill KK, Lowery KS, Van Ert M, Pearson T, Schupp J, Kenefic L, Beaudry J, Hofstadler SA, Jackson PJ, Keim P (2008) Single nucleotide polymorphism typing of Bacillus anthracis from Sverdlovsk tissue. Emerg Infect Dis 14(4):653–656

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Pearson T, Busch JD, Ravel J, Read TD, Rhoton SD, U’ren JM, Simonson TS, Kachur SM, Leadem RR, Cardon ML, Van Ert MN, Huynh LY, Fraser CM, Keim P (2004) Phylogenetic discovery bias in Bacillus anthracis using single-nucleotide polymorphisms from whole-genome sequencing. Proc Natl Acad Sci U S A 101(37):13536–13541

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Read TD, Salzberg SL, Pop M, Shumway M, Umayam L, Jiang L, Holtzapple E, Busch JD, Smith KL, Schupp JM, Solomon D, Keim P, Fraser CM (2002) Comparative genome sequencing for discovery of novel polymorphisms in Bacillus anthracis. Science 296(5575):2028–2033

    Article  CAS  PubMed  Google Scholar 

  21. Price LB, Hugh-Jones M, Jackson PJ, Keim P (1999) Genetic diversity in the protective antigen gene of Bacillus anthracis. J Bacteriol 181(8):2358–2362

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Jackson PJ, Hill KK, Laker MT, Ticknor LO, Keim P (1999) Genetic comparison of Bacillus anthracis and its close relatives using amplified fragment length polymorphism and polymerase chain reaction analysis. J Appl Microbiol 87(2):263–269

    Article  CAS  PubMed  Google Scholar 

  23. Harrell LJ, Andersen GL, Wilson KH (1995) Genetic variability of Bacillus anthracis and related species. J Clin Microbiol 33(7):1847–1850

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Keim P, Kalif A, Schupp J, Hill K, Travis SE, Richmond K, Adair DM, Hugh-Jones M, Kuske CR, Jackson P (1997) Molecular evolution and diversity in Bacillus anthracis as detected by amplified fragment length polymorphism markers. J Bacteriol 179(3):818–824

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Joseph SJ, Read TD (2010) Bacterial population genomics and infectious disease diagnostics. Trends Biotechnol 28(12):611–618

    Article  CAS  PubMed  Google Scholar 

  26. Cummings CA, Bormann Chung CA, Fang R, Barker M, Brzoska PM, Williamson P, Beaudry JA, Matthews M, Schupp JM, Wagner DM (2009) Whole-genome typing of Bacillus anthracis isolates by next-generation sequencing accurately and rapidly identifies strain-specific diagnostic polymorphisms. Forensic Sci Int Genet Suppl Ser 2(1):300–301

    Article  Google Scholar 

  27. Beyer W, Bellan S, Eberle G, Ganz HH, Getz WM, Haumacher R, Hilss KA, Kilian W, Lazak J, Turner WC, Turnbull PC (2012) Distribution and molecular evolution of Bacillus anthracis genotypes in Namibia. PLoS Negl Trop Dis 6(3):e1534

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Le Flèche P, Hauck Y, Onteniente L, Prieur A, Denoeud F, Ramisse V, Sylvestre P, Benson G, Ramisse F, Vergnaud G (2001) A tandem repeats database for bacterial genomes: application to the genotyping of Yersinia pestis and Bacillus anthracis. BMC Microbiol 1(1):2

    Article  PubMed  PubMed Central  Google Scholar 

  29. 闫明媚, 兰德松, 赵凤菊, 顾贵波, 魏澍 (2012) 辽宁省牛炭疽疫情诊断报告. 现代畜牧兽医 (4):21–22

  30. Pennisi E (2010) Genomics. Semiconductors inspire new sequencing technologies. Science 327(5970):1190

    Article  CAS  PubMed  Google Scholar 

  31. Zerbino DR, Birney E (2008) Velvet: algorithms for de novo short read assembly using de Bruijn graphs. Genome Res 18(5):821–829

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Li R, Li Y, Kristiansen K, Wang J (2008) SOAP: short oligonucleotide alignment program. Bioinformatics 24(5):713–714

    Article  CAS  PubMed  Google Scholar 

  33. Aziz RK, Bartels D, Best AA, DeJongh M, Disz T, Edwards RA, Formsma K, Gerdes S, Glass EM, Kubal M, Meyer F, Olsen GJ, Olson R, Osterman AL, Overbeek RA, McNeil LK, Paarmann D, Paczian T, Parrello B, Pusch GD, Reich C, Stevens R, Vassieva O, Vonstein V, Wilke A, Zagnitko O (2008) The RAST Server: rapid annotations using subsystems technology. BMC Genomics 9(1):75

    Article  PubMed  PubMed Central  Google Scholar 

  34. Lagesen K, Hallin P, Rødland EA, Stærfeldt H-H, Rognes T, Ussery DW (2007) RNAmmer: consistent and rapid annotation of ribosomal RNA genes. Nucleic Acids Res 35(9):3100–3108

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Simonson TS, Okinaka RT, Wang B, Easterday WR, Huynh L, U’Ren JM, Dukerich M, Zanecki SR, Kenefic LJ, Beaudry J, Schupp JM, Pearson T, Wagner DM, Hoffmaster A, Ravel J, Keim P (2009) Bacillus anthracis in China and its relationship to worldwide lineages. BMC Microbiol 9(1):71

    Article  PubMed  PubMed Central  Google Scholar 

  36. Arnold K, Bordoli L, Kopp J, Schwede T (2006) The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling. Bioinformatics 22(2):195–201

    Article  CAS  PubMed  Google Scholar 

Download references

Conflict of interest

The authors declare that they have no conflict of interest.

Author information

Authors and Affiliations

Authors

Corresponding authors

Correspondence to G. Gu or Y. Tong.

Additional information

S. Li, X. An, and Y. Huang contributed equally to this work.

Electronic supplementary material

Below are the links to the electronic supplementary material.

ESM 1

(XLS 19 kb)

ESM 2

(XLSX 17 kb)

ESM 3

(XLSX 23 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Li, S., An, X., Huang, Y. et al. Source tracking of an anthrax outbreak in northeastern China using complete genome analysis and MLVA genotyping. Eur J Clin Microbiol Infect Dis 34, 89–100 (2015). https://doi.org/10.1007/s10096-014-2195-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10096-014-2195-7

Keywords

Navigation