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Applied and Environmental Microbiology, February 2006, p. 1295-1301, Vol. 72, No. 2
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.2.1295-1301.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Strategy for Identification of Bacillus cereus and Bacillus thuringiensis Strains Closely Related to Bacillus anthracis

Daniele Daffonchio,^1* Noura Raddadi,¹ Maya Merabishvili,² Ameur Cherif,^1,3 Lorenzo Carmagnola,¹ Lorenzo Brusetti,¹ Aurora Rizzi,¹ Nina Chanishvili,² Paolo Visca,⁴ Richard Sharp,⁵ and Sara Borin¹

Dipartimento di Scienze e Tecnologie Alimentari e Microbiologiche, Università Degli Studi di Milano, 20133 Milan, Italy,¹ G. Eliava Institute of Bacteriophage, Microbiology and Virology, 0160 Tbilisi, Georgia,² Laboratoire de Microbiologie, Département de Biologie, Faculté des Sciences de Tunis, 2092 Tunis, Tunisia,³ Istituto Nazionale per le Malattie Infettive I.R.C.C.S. ‘Lazzaro Spallanzani,’ 00142 Rome, Italy,⁴ Health Protection Agency, Porton Down, Salisbury SP4 OJG, United Kingdom⁵

Received 4 August 2005/ Accepted 8 November 2005

Bacillus cereus strains that are genetically closely related to B. anthracis can display anthrax-like virulence traits (A. R. Hoffmaster et al., Proc. Natl. Acad. Sci. USA 101:8449-8454, 2004). Hence, approaches that rapidly identify these "near neighbors" are of great interest for the study of B. anthracis virulence mechanisms, as well as to prevent the use of such strains for B. anthracis-based bioweapon development. Here, a strategy is proposed for the identification of near neighbors of B. anthracis based on single nucleotide polymorphisms (SNP) in the 16S-23S rRNA intergenic spacer (ITS) containing tRNA genes, characteristic of B. anthracis. By using restriction site insertion-PCR (RSI-PCR) the presence of two SNP typical of B. anthracis was screened in 126 B. cereus group strains of different origin. Two B. cereus strains and one B. thuringiensis strain showed RSI-PCR profiles identical to that of B. anthracis. The sequencing of the entire ITS containing tRNA genes revealed two of the strains to be identical to B. anthracis. The strict relationship with B. anthracis was confirmed by multilocus sequence typing (MLST) of four other independent loci: cerA, plcR, AC-390, and SG-749. The relationship to B. anthracis of the three strains described by MLST was comparable and even higher to that of four B. cereus strains associated with periodontitis in humans and previously reported as the closest known strains to B. anthracis. SNP in ITS containing tRNA genes combined with RSI-PCR provide a very efficient tool for the identification of strains closely related to B. anthracis.

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* Corresponding author. Mailing address: Dipartimento di Scienze e Tecnologie Alimentari e Microbiologiche, Università degli Studi di Milano, via Celoria 2, 20133 Milan, Italy. Phone: 39-0250316730. Fax: 39-0250316694. E-mail: daniele.daffonchio{at}unimi.it.

Supplemental material for this article may be found at http://aem.asm.org/.

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Applied and Environmental Microbiology, February 2006, p. 1295-1301, Vol. 72, No. 2
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.2.1295-1301.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.