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MLVA and SNP analysis identified a unique genetic cluster in Bulgarian Bacillus anthracis strains

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Abstract

A collection of 40 Bacillus anthracis strains mostly isolated from soil in Bulgaria between 1960 and 1980 were investigated. All strains were proven to be B. anthracis by culture and amplification of a B. anthracis-specific chromosomal marker. PCR demonstrated that in nine strains both virulence plasmids (pX01+/pX02+) and in four strains only one plasmid (pX02+) were present, whereas the majority of strains (n = 27) lacked both plasmids (pX01-/pX02-). Multi-locus-variable number of tandem repeat-analysis (MLVA) using 15 markers differentiated three genotypes. Comparison with typing data of more than 1,000 different B. anthracis strains revealed that Bulgarian genotypes affiliated with the A1.a cluster and form their own unique cluster different from clusters containing strains isolated in geographical proximity, e.g., Turkey, Georgia, Hungary, Albania or Italy. In addition, a new allele of one marker (vrrC2) was identified. Canonical single nucleotide polymorphisms analysis allocated 31 Bulgarian strains into the A.Br.008/009 and nine strains into the A.Br.WNA group, which is the first description of B. anthracis strains of the A.Br.WNA group on the Eurasian continent.

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References

  1. WHO (2008) Anthrax in humans and animals. WHO library cataloguing. http://www.who.int/csr/resources/publications/AnthraxGuidelines2008/en/index.html. Accessed 11 December 2010

  2. Greene CM, Reefhuis J, Tan C, Fiore AE, Goldstein S, Beach MJ, Redd SC, Valiante D, Burr G, Buehler J, Pinner RW, Bresnitz E, Bell BP (2002) Epidemiologic investigations of bioterrorism-related anthrax, New Jersey, 2001. Emerg Infect Dis 8:1048–1055

    PubMed  Google Scholar 

  3. Holtz TH, Ackelsberg J, Kool JL, Rosselli R, Marfin A, Matte T, Beatrice ST, Heller MB, Hewett D, Moskin LC, Bunning ML, Layton M (2003) Isolated case of bioterrorism-related inhalational anthrax, New York City, 2001. Emerg Infect Dis 9:689–696

    PubMed  Google Scholar 

  4. Jernigan JA, Stephens DS, Ashford DA, Omenaca C, Topiel MS, Galbraith M, Tapper M, Fisk TL, Zaki S, Popovic T, Meyer RF, Quinn CP, Harper SA, Fridkin SK, Sejvar JJ, Shepard CW, McConnell M, Guarner J, Shieh WJ, Malecki JM, Gerberding JL, Hughes JM, Perkins BA (2001) Bioterrorism-related inhalational anthrax: the first 10 cases reported in the United States. Emerg Infect Dis 7:933–944

    Article  PubMed  CAS  Google Scholar 

  5. Keim P, Smith KL, Keys C, Takahashi H, Kurata T, Kaufmann A (2001) Molecular investigation of the Aum Shinrikyo anthrax release in Kameido, Japan. J Clin Microbiol 39:4566–4567

    Article  PubMed  CAS  Google Scholar 

  6. Takahashi H, Keim P, Kaufmann AF, Keys C, Smith KL, Taniguchi K, Inouye S, Kurata T (2004) Bacillus anthracis incident, Kameido, Tokyo, 1993. Emerg Infect Dis 10:117–120

    PubMed  Google Scholar 

  7. Van Ert MN, Easterday WR, Simonson TS, U'Ren JM, Pearson T, Kenefic LJ, Busch JD, Huynh LY, Dukerich M, Trim CB, Beaudry J, Welty-Bernard A, Read T, Fraser CM, Ravel J, Keim P (2007) Strain-specific single-nucleotide polymorphism assays for the Bacillus anthracis Ames strain. J Clin Microbiol 45:47–53

    Article  PubMed  Google Scholar 

  8. Merabishvili M, Natidze M, Rigvava S, Brusetti L, Raddadi N, Borin S, Chanishvili N, Tediashvili M, Sharp R, Barbeschi M, Visca P, Daffonchio D (2006) Diversity of Bacillus anthracis strains in Georgia and of vaccine strains from the former Soviet Union. Appl Environ Microbiol 72:5631–5636

    Article  PubMed  CAS  Google Scholar 

  9. 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:e461

    Article  PubMed  Google Scholar 

  10. 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 48:2821–2829

    Article  PubMed  CAS  Google Scholar 

  11. Aikembayev AM, Lukhnova L, Temiraliyeva G, Meka-Mechenko T, Pazylov Y, Zakaryan S, Denissov G, Easterday WR, Van Ert MN, Keim P, Francesconi SC, Blackburn JK, Hugh-Jones M, Hadfield T (2010) Historical distribution and molecular diversity of Bacillus anthracis, Kazakhstan. Emerg Infect Dis 16:789–796

    PubMed  CAS  Google Scholar 

  12. Antwerpen M, Zimmermann P, Frangoulidis D, Bewley K, Meyer H (2008) Real-time PCR system targeting a chromosomal marker specific for Bacillus anthracis. Mol Cell Probes 22:313–315

    Article  PubMed  CAS  Google Scholar 

  13. Vergnaud G, Pourcel C (2006) Multiple Locus VNTR (Variable Number of Tandem Repeat) Analysis (MLVA). In: E Stackebrandt (ed) Molecular identification, systematics and population structure of prokaryotes. Springer, pp 83–104

  14. 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 179:818–824

    PubMed  CAS  Google Scholar 

  15. Kenefic LJ, Pearson T, Okinaka RT, Schupp JM, Wagner DM, Hoffmaster AR, Trim CB, Chung WK, Beaudry JA, Jiang L, Gajer P, Foster JT, Mead JI, Ravel J, Keim P (2009) Pre-Columbian origins for North American anthrax. PLoS ONE 4:e4813

    Article  PubMed  Google Scholar 

  16. Fasanella A, Van Ert M, Altamura SA, Garofolo G, Buonavoglia C, Leori G, Huynh L, Zanecki S, Keim P (2005) Molecular diversity of Bacillus anthracis in Italy. J Clin Microbiol 43:3398–3401

    Article  PubMed  CAS  Google Scholar 

  17. 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 9:71

    Article  PubMed  Google Scholar 

  18. 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 4:205–213

    Article  PubMed  CAS  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 USA 101:13536–13541

    Article  PubMed  CAS  Google Scholar 

  20. Keim P, Price L, Klevytska A, Smith K, Schupp J, Okinaka R, Jackson P, Hugh-Jones M (2000) Multiple-locus variable-number tandem repeat analysis reveals genetic relationships within Bacillus anthracis. J Bacteriol 182:2928–2936

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  Google Scholar 

  22. Lista F, Faggioni G, Valjevac S, Ciammaruconi A, Vaissaire J, le Doujet C, Gorge O, De Santis R, Carattoli A, Fasanella A, Orsini F, D'Amelio R, Pourcel C, Cassone A, Vergnaud G (2006) Genotyping of Bacillus anthracis strains based on automated capillary 25-loci multiple locus variable-number tandem repeats analysis. BMC Microbiol 6:33

    Article  PubMed  Google Scholar 

  23. Wattiau P, Klee SR, Fretin D, Van Hessche M, Ménart M, Franz T, Chasseur C, Butaye P, Imberechts H (2008) Occurrence and genetic diversity of Bacillus anthracis strains isolated in an active wool-cleaning factory. Appl Environ Microbiol 74:4005–4011

    Article  PubMed  CAS  Google Scholar 

  24. Pilo P, Perreten V, Frey J (2008) Molecular epidemiology of Bacillus anthracis: determining the correct origin. Appl Environ Microbiol 74:2928–2931

    Article  PubMed  CAS  Google Scholar 

  25. Marston CK, Hoffmaster AR, Wilson KE, Bragg SL, Plikaytis B, Brachman P, Johnson S, Kaufmann AF, Popovic T (2005) Effects of long-term storage on plasmid stability in Bacillus anthracis. Appl Environ Microbiol 71:7778–7780

    Article  PubMed  CAS  Google Scholar 

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Acknowledgment

We gratefully acknowledge Claudia Kahlhofer and Linda Dobrzykowski for excellent technical assistance. We also thank the unknown reviewers for helpful comments.

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Correspondence to M. Antwerpen.

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Antwerpen, M., Ilin, D., Georgieva, E. et al. MLVA and SNP analysis identified a unique genetic cluster in Bulgarian Bacillus anthracis strains. Eur J Clin Microbiol Infect Dis 30, 923–930 (2011). https://doi.org/10.1007/s10096-011-1177-2

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  • DOI: https://doi.org/10.1007/s10096-011-1177-2

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