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.
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References
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
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
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
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
Mock M, Fouet A (2001) Anthrax. Annu Rev Microbiol 55(1):647–671
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
Turnbull PC (1999) Definitive identification of Bacillus anthracis—a review. J Appl Microbiol 87(2):237–240
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
Rasko DA, Altherr MR, Han CS, Ravel J (2005) Genomics of the Bacillus cereus group of organisms. FEMS Microbiol Rev 29(2):303–329
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
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
Eitzen EM (1917) Use of biological weapons. Med Asp Chem Biol Warf 41(2):450
Pile JC, Malone JD, Eitzen EM, Friedlander AM (1998) Anthrax as a potential biological warfare agent. Arch Intern Med 158(5):429–434
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
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
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
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
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
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
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
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
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
Harrell LJ, Andersen GL, Wilson KH (1995) Genetic variability of Bacillus anthracis and related species. J Clin Microbiol 33(7):1847–1850
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
Joseph SJ, Read TD (2010) Bacterial population genomics and infectious disease diagnostics. Trends Biotechnol 28(12):611–618
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
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
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
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
Pennisi E (2010) Genomics. Semiconductors inspire new sequencing technologies. Science 327(5970):1190
Zerbino DR, Birney E (2008) Velvet: algorithms for de novo short read assembly using de Bruijn graphs. Genome Res 18(5):821–829
Li R, Li Y, Kristiansen K, Wang J (2008) SOAP: short oligonucleotide alignment program. Bioinformatics 24(5):713–714
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
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
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
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
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S. Li, X. An, and Y. Huang contributed equally to this work.
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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
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DOI: https://doi.org/10.1007/s10096-014-2195-7