Generation of a Specific Marker to Discriminate Bacillus anthracis from Other Bacteria of the Bacillus cereus Group

  • Kim, Hyoung-Tai (Division of Molecular and Life Sciences, Hanyang University) ;
  • Seo, Gwi-Moon (Division of Molecular and Life Sciences, Hanyang University) ;
  • Jung, Kyoung-Hwa (Division of Molecular and Life Sciences, Hanyang University) ;
  • Kim, Seong-Joo (Division of Molecular and Life Sciences, Hanyang University) ;
  • Kim, Jee-Cheon (Division of Molecular and Life Sciences, Hanyang University) ;
  • Oh, Kwang-Geun (Department of Bioprocess Technology, BioPolytechnic College) ;
  • Koo, Bon-Sung (Microbial Genetics Division, National Institute of Agricultural Biotechnology, RDA) ;
  • Chai, Young-Gyu (Division of Molecular and Life Sciences, Hanyang University)
  • Published : 2007.05.31

Abstract

Bacillus anthracis is a soil pathogen capable of causing anthrax that is closely related to several environmental species, including B. cereus, B. mycoides, and B. thuringiensis. DNA homology studies showed that B. anthracis, B. cereus, B. mycoides, and B. thuringiensis are closely related, with a high sequence homology. To establish a method to specifically detect B. anthracis in situations such as environmental contamination, we initially performed RAPD-PCR with a 10-mer random primer and confirmed the presence of specific PCR bands only in B. anthracis species. One region specific for B. anthracis was cloned and sequenced, and an internal primer set was designed to amplify a 241-bp DNA fragment within the sequenced region. The PCR system involving these specific primer sets has practical applications. Using lyses methods to prepare the samples for PCR, it was possible to quickly amplify the 241-bp DNA segment from samples containing only a few bacteria. Thus, the PCR detection method developed in this study is expected to facilitate the monitoring of environmental B. anthracis contamination.

Keywords

References

  1. Bavykin, S. G., Y. P. Lysov, V. Zakhariev, J. J. Kelly, J. Jackman, D. A. Stahl, and A. Cherni. 2004. Use of 16S rRNA, 23S rRNA, and gyrB gene sequence analysis to determine phylogenetic relationships of Bacillus cereus group microorganisms. J. Clin. Microbiol. 42: 3711-3730 https://doi.org/10.1128/JCM.42.8.3711-3730.2004
  2. Bode, E., W. Hurtle, and D. Norwood. 2004. Real-time PCR assay for a unique chromosomal sequence of Bacillus anthracis. J. Clin. Microbiol. 42: 5825-5831 https://doi.org/10.1128/JCM.42.12.5825-5831.2004
  3. Broussard, L. A. 2001. Biological agents: Weapons of warfare and bioterrorism. Mol. Diagn. 6: 323-333 https://doi.org/10.2165/00066982-200106040-00013
  4. Brousseau, R., A. Saintonge, G. Prefontaine, L. Masson, and J. Cabana. 1993. Arbitrary primer polymerase chainreaction, a powerful method to identify Bacillus thuringiensis serovars and strains. Appl. Environ. Microbiol. 59: 114- 119
  5. Bruno, J. G. and H. Yu. 1996. Immunomagneticelectrochemiluminescent detection of Bacillus anthracis spores in soil matrices. Appl. Environ. Microbiol. 62: 3474- 3476
  6. Castanha, E. R., R. R. Swiger, B. Senior, A. Fox, L. N. Waller, and K. F. Fox. 2006. Strain discrimination among B. anthracis and related organisms by characterization of bclA polymorphisms using PCR coupled with agarose gel or microchannel fluidics electrophoresis. J. Microbiol. Methods 64: 27-45 https://doi.org/10.1016/j.mimet.2005.04.032
  7. Chen, M. L. and H. Y. Tsen. 2002. Discrimination of Bacillus cereus and Bacillus thuringiensis with 16S rRNA and gyrB gene based PCR primers and sequencing of their annealing sites. J. Appl. Microbiol. 92: 912-919 https://doi.org/10.1046/j.1365-2672.2002.01606.x
  8. Cherif, A., S. Borin, A. Rizzi, H. Ouzari, A. Boudabous, and D. Daffonchio. 2003. Bacillus anthracis diverges from related clades of the Bacillus cereus group in 16S-23S ribosomal DNA intergenic transcribed spacers containing tRNA genes. Appl. Environ. Microbiol. 69: 33-40 https://doi.org/10.1128/AEM.69.1.33-40.2003
  9. Cherif, A., L. Brusetti, S. Borin, A. Rizzi, A. Boudabous, H. Khyami-Horani, and D. Daffonchio. 2003. Genetic relationship in the 'Bacillus cereus group' by rep-PCR fingerprinting and sequencing of a Bacillus anthracis-specific rep-PCR fragment. J. Appl. Microbiol. 94: 1108-1119 https://doi.org/10.1046/j.1365-2672.2003.01945.x
  10. DelVecchio, V. G., J. P. Connolly, T. G. Alefantis, A. Walz, M. A. Quan, G. Patra, J. M. Ashton, J. T. Whittington, R. D. Chafin, X. D. Liang, P. Grewal, A. S. Khan, and C. V. Mujer. 2006. Proteomic profiling and identification of immunodominant spore antigens of Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis. Appl. Environ. Microbiol. 72: 6355-6363 https://doi.org/10.1128/AEM.00455-06
  11. Edgar, R., M. McKinstry, J. Hwang, A. B. Oppenheim, R. A. Fekete, G. Giulian, C. Merril, K. Nagashima, and S. Adhya. 2006. High-sensitivity bacterial detection using biotin-tagged phage and quantum-dot nanocomplexes. Proc. Natl. Acad. Sci. USA 103: 4841-4845
  12. Guarise, C., L. Pasquato, V. De Filippis, and P. Scrimin. 2006. Gold nanoparticles-based protease assay. Proc. Natl. Acad. Sci. USA 103: 3978-3982
  13. Ha, C.-G., J.-K. Cho, C.-H. Lee, Y.-G. Chai, Y.-A. Ha, and S.-H. Shin. 2006. Cholesterol lowering effect of Lactobacillus plantarum isolated from human feces. J. Microbiol. Biotechnol. 16: 1201-1209
  14. Ha, C.-G., J.-K. Cho, Y.-G. Chai, Y.-A. Ha, and S.-H. Shin. 2006. Purification and characterization of bile salt hydrolase from Lactobacillus plantarum CK 102. J. Microbiol. Biotechnol. 16: 1047-1052
  15. Hansen, B. M., T. D. Leser, and N. B. Hendriksen. 2001. Polymerase chain reaction assay for the detection of Bacillus cereus group cells. FEMS Microbiol. Lett. 202: 209-213 https://doi.org/10.1111/j.1574-6968.2001.tb10805.x
  16. Jung, S.-Y., J.-H. Lee, Y.-G. Chai, and S.-J. Kim. 2005. Monitoring of microorganisms added into oil-contaminated microenvironments by terminal-restriction fragment length polymorphism analysis. J. Microbiol. Biotechnol. 15: 1170- 1177
  17. Klee, S. R., H. Nattermann, S. Becker, M. Urban-Schriefer, T. Franz, D. Jacob, and B. Appel. 2006. Evaluation of different methods to discriminate Bacillus anthracis from other bacteria of the Bacillus cereus group. J. Appl. Microbiol. 100: 673-681 https://doi.org/10.1111/j.1365-2672.2006.02809.x
  18. La Duc, M. T., M. Satomi, N. Agata, and K. Venkateswaran. 2004. GyrB as a phylogenetic discriminator for members of the Bacillus anthracis-cereus-thuringiensis group. J. Microbiol. Methods 56: 383-394 https://doi.org/10.1016/j.mimet.2003.11.004
  19. Levine, S. A., Y. W. Tang, and Z. H. Pei. 2005. Recent advances in the rapid detection of Bacillus anthracis. Rev. Med. Microbiol. 16: 125-133 https://doi.org/10.1097/01.revmedmi.0000184744.83303.9a
  20. Park, T.-J., J.-P. Park, G.-M. Seo, Y.-G. Chai, and S.-Y. Lee. 2006. Rapid and accurate detection of Bacillus anthracis spores using peptide-quantum dot conjugates. J. Microbiol. Biotechnol. 16: 1713-1719
  21. Radnedge, L., P. G. Agron, K. K. Hill, P. J. Jackson, L. O. Ticknor, P. Keim, and G. L. Andersen. 2003. Genome differences that distinguish Bacillus anthracis from Bacillus cereus and Bacillus thuringiensis. Appl. Environ. Microbiol. 69: 2755-2764 https://doi.org/10.1128/AEM.69.5.2755-2764.2003
  22. Ramisse, V., G. Patra, H. Garrigue, J. L. Guesdon, and M. Mock. 1996. Identification and characterization of Bacillus anthracis by multiplex PCR analysis of sequences on plasmids pXO1 and pXO2 and chromosomal DNA. FEMS Microbiol. Lett. 145: 9-16 https://doi.org/10.1111/j.1574-6968.1996.tb08548.x
  23. Ramisse, V., G. Patra, J. Vaissaire, and M. Mock. 1999. The Ba813 chromosomal DNA sequence effectively traces the whole Bacillus anthracis community. J. Appl. Microbiol. 87: 224-228 https://doi.org/10.1046/j.1365-2672.1999.00874.x
  24. Ryu, C., K. Lee, C. Yoo, W. K. Seong, and H. B. Oh. 2003. Sensitive and rapid quantitative detection of anthrax spores isolated from soil samples by real-time PCR. Microbiol. Immunol. 47: 693-699 https://doi.org/10.1111/j.1348-0421.2003.tb03434.x
  25. Seo, G. M., S. J. Kim, and Y. G. Chai. 2004. Rapid profiling of the infection of Bacillus anthracis on human macrophages using SELDI-TOF mass spectroscopy. Biochem. Biophys. Res. Commun. 325: 1236-1239 https://doi.org/10.1016/j.bbrc.2004.10.146
  26. Seo, G. M., S. J. Kim, J. C. Kim, D. H. Nam, M. Y. Yoon, B. S. Koo, and Y. G. Chai. 2004. Targeting of Bacillus anthracis interaction factors for human macrophages using two-dimensional gel electrophoresis. Biochem. Biophys. Res. Commun. 322: 854-859 https://doi.org/10.1016/j.bbrc.2004.07.190
  27. Turnbull, P. C. B., R. A. Hutson, M. J. Ward, M. N. Jones, C. P. Quinn, N. J. Finnie, C. J. Duggleby, J. M. Kramer, and J. Melling. 1992. Bacillus-anthracis but not always anthrax. J. Appl. Bacteriol. 72: 21-28
  28. Zhang, X. Y., M. A. Young, O. Lyandres, and R. P. Van Duyne. 2005. Rapid detection of an anthrax biomarker by surface-enhanced Raman spectroscopy. J. Amer. Chem. Soc. 127: 4484-4489 https://doi.org/10.1021/ja043623b