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