JOURNAL OF CLINICAL MICROBIOLOGY, Apr. 2006, p. 1606–1607 0095-1137/06/$08.00⫹0 doi:10.1128/JCM.44.4.1606–1607.2006 Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Vol. 44, No. 4
Some Bacillus thuringiensis Strains Share rpoB Nucleotide Polymorphisms Also Present in Bacillus anthracis Identification of Bacillus anthracis is considerably difficult because of its very high phenotypic and genotypic similarity to other members of the Bacillus cereus group. Differentiation methods based on morphological and phenotypic characteristics are time-consuming, and false results may be obtained for atypical strains. On the other hand molecular discrimination based on the presence of two B. anthracis virulence plasmids, pXO1 and pXO2, is not sufficient because plasmids can be lost or transferred to other bacilli. Therefore, several chromosomal markers have been investigated and applied (1, 8). In 2001 Qi et al. (7) described single nucleotide polymorphisms (SNPs) in the rpoB gene and their usefulness for B. anthracis identification. Since then, several articles describing various molecular methods for rpoB sequence-based detection of B. anthracis have been published (for example see references 2 and 9). We conducted studies of single-strand conformation polymorphisms (SSCPs) of the rpoB gene in a large collection of B. cereus group strains. Surprisingly, we found that the nucleotide sequence of the rpoB gene fragment containing the marker SNPs of two reference strains of Bacillus thuringiensis was identical to that of the homologous region in B. anthracis. Therefore, rpoB gene-based tools could not distinguish these strains from B. anthracis, thus resulting in false-positive anthrax identification. We tested 95 samples of total genomic DNA of Bacillus species belonging to the B. cereus group (B. anthracis, B. thuringiensis, B. cereus, B. weihenstephanensis, B. mycoides, and B. pseudomycoides) using the multitemperature-SSCP technique, which has an extended capacity to detect SNPs (6). Primers rpoB1f (5⬘-GGTGATAACGAATACTTAAGC-3⬘) and rpoB1r (5⬘-AATGCGATCAAGTGTACGACG-3⬘) were used to amplify a 321-bp fragment of the rpoB gene encompassing marker SNPs described by Qi et al. (7). Amplification was performed using AmpliTaq Gold DNA polymerase (Applied Biosystems). PCR products were denatured as previously described (4) and loaded onto an 8% polyacrylamide gel containing 3.75% glycerol. The multitemperature-SSCP assay was carried out in Trisbuffered EDTA in DNA Pointer (Kucharczyk TE, Poland) with 40 W of constant power. The gel temperatures were 35°C, 20°C, and 5°C, each stage lasting 45 min. Gels were silver stained, scanned, and analyzed using GelScan software version 1.3 (Kucharczyk TE). The multitemperature-SSCP analysis of the rpoB marker revealed indistinguishable profiles for all B. anthracis strains tested whereas most of the nonanthrax bacilli generated apparently different profiles. However, two B. thuringiensis strains tested, HD146 (serovar dermstadiensis) and HD868 (serovar tochigiensis) (10), exhibited the same profile as B. anthracis did (Fig. 1). We performed DNA sequencing of these amplicons using an automated fluorescent 377 DNA sequencer and BigDye Terminator v3.1 (Applied Biosystems) in accordance with the manufacturer’s instructions. Each strand of the analyzed amplicons was sequenced separately using primer rpoB1f or rpoB1r. The final sequence of the rpoB core region covering the marker SNPs was determined by comparison of data from the two strands. Results obtained indicated that rpoB core regions of B. thuringiensis HD146 and HD868 were indistin-
guishable from each other as well from the sequence reported by Qi et al. (7) for B. anthracis. To confirm that PCR templates HD146 and HD868 were obtained from nonanthrax bacilli, we conducted the SG-749 PCR-restriction fragment length polymorphism (RFLP) assay described by Daffonchio et al. (1). The RFLP patterns of HD146 and HD868 were evidently dissimilar to that of the reference strain Sterne 34F2, indicating that these samples contained non-B. anthracis DNA. Taken together, results obtained by DNA sequencing and the PCR-RFLP assay indicate that we found rarely occurring B. thuringiensis strains possessing B. anthracis-specific SNPs in the rpoB gene. To our knowledge, such strains have not been reported to date. Interestingly, in 2002, Ellerbrok et al. (2) found that some strains of B. cereus can give positive signals in rpoB targeted real-time PCR over 10 cycles later than B. anthracis can. Their results showed that B. cereus may possess an rpoB gene having a sequence closely resembling the sequence present in B. anthracis. However, the rpoB sequence of the aforementioned B. cereus strains was not determined. In 2005 Elzi et al. (3) described a B. cereus isolate possessing three of the four B. anthracis characteristic SNPs in rpoB. We found two B. thuringiensis strains with nucleotide polymorphisms in the rpoB gene identical to those of B. anthracis. The findings support the concept that B. anthracis, B. cereus, and B. thuringiensis belong to the same species and that what distinguishes them functionally are mostly genes carried on plasmids (5). Nevertheless, plasmids might be lost or transferred to other bacilli. Keeping in mind the risk of bioterrorism as well as the need for monitoring the occurrence of B. anthracis in the environment, identification methods independent of the presence of plasmids are essential. But these results raise the question of whether rpoB can be considered a reliable marker for specific anthrax identification.
FIG. 1. A. Multitemperature-SSCP analysis of the rpoB gene. Lane 1, B. anthracis Sterne 34F2; lane 2, B. thuringiensis HD146; lane 3, B. thuringiensis HD868; lanes 4 to 12, other B. thuringiensis strains tested. B. SG-749 RFLP patterns of DNA separated on a polyacrylamide gel and silver stained. Lane 1, 100-bp ladder; lanes 2 and 5, B. anthracis Sterne 34F2; lane 3, B. thuringiensis HD146; lane 4, B. thuringiensis HD868. 1606
VOL. 44, 2006
LETTERS TO THE EDITOR
This study was supported by a research grant, 2 PO5D 07529, from MSIST. D.D. was supported by ISPELS within the project B74/MDL/02. ´wie˛cicka from the University of Białystok, Białystok, We thank I. S Poland, for providing B. thuringiensis strains and P. E. Granum from The Norwegian School of Veterinary Science, Oslo, Norway, for providing B. weihenstephanensis strains. REFERENCES 1. Daffonchio, D., S. Robin, G. Frova, R. Gallo, E. Mori, R. Fani, and C. Sorlini. 1999. A randomly amplified polymorphic DNA marker specific for the Bacillus cereus group is diagnostic for Bacillus anthracis. Appl. Environ. Microbiol. 65:1298–1303. 2. Ellerbrok, H., H. Nattermann, M. Ozel, L. Beutin, B. Appel, and G. Pauli. 2002. Rapid and sensitive identification of pathogenic and apathogenic Bacillus anthracis by real-time PCR. FEMS Microbiol. Lett. 214:51–59. 3. Elzi, M. V., K. Mallard, S. Drozd, and T. Bodmer. 2005. Polyphasic approach for identufying Bacillus spp. J. Clin. Microbiol. 43:1010. 4. Gierczyn ´ ski, R., S. Kałuz˙ewski, A. Rakin, M. Jagielski, A. Zasada, A. Jakubczak, B. Borkowska-Opacka, and W. Rastawicki. 2004. Intriguing diversity of Bacillus anthracis in eastern Poland—the molecular echoes of the past outbreaks. FEMS Microbiol. Lett. 239:235–240. 5. Helgason, E., O. A. Økstad, D. A. Caugant, H. A. Johansen, A. Fouet, M. Mock, I. Hegna, and A.-B. Kolstø. 2000. Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis—one species on the basic of genetic evidence. Appl. Environ. Microbiol. 66:2627–2630. 6. Kaczanowski, R., L. Trzeciak, and K. Kucharczyk. 2001. Multitemperature single-strand conformation polymorphism. Electrophoresis 22:3539–3545. 7. Qi, Y., G. Patra, X. Liang, L. E. Williams, S. Rose, R. J. Redkar, and V. G. DelVecchio. 2001. Utilization of the rpoB gene as a specific chromosomal marker for real-time PCR detection of Bacillus anthracis. Appl. Environ. Microbiol. 67:3720–3727. 8. Radnedge, L., P. G. Agron, K. K. Hill, P. J. Jackson, L. O. Ticknor, P. Keim,
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and G. L. Andersen. 2003. Genome differences that distinguish Bacillus anthracis from Bacillus cereus and Bacillus thuringiensis. Appl. Environ. Microbiol. 69:2755–2764. 9. Wahab, T., S. Hjalmarsson, R. Wollin, and L. Engstrand. 2005. Pyrosequencing Bacillus anthracis. Emerg. Infect. Dis. 11:1527–1531. 10. Zeigler, D. R. 1999. Bacillus Genetic Stock Center catalog of strains. Part 2: Bacillus thuringiensis and Bacillus cereus, 7th ed. Bacillus Genetic Stock Center, Columbus, Ohio. [Online.] http://www.bgsc.org/Catalogs/Catpart2.pdf.
Aleksandra Anna Zasada* Rafaèł Gierczyn ´ ski Department of Bacteriology National Institute of Hygiene Warsaw, Poland Noura Raddadi Daniele Daffonchio Dipartimento di Scienze e Tecnologie Alimentari a Microbiologiche Universita ` degli Studi Milano, Italy Marek Jagielski Department of Bacteriology National Institute of Hygiene Warsaw, Poland *Phone: 48 22 54 21 244 Fax: 48 22 54 21 307 E-mail:
[email protected] 