Characterization of a repetitive element polymorphism- polymerase chain reaction chromosomal marker that discriminates Bacillus anthracis from related species A. Cherif 1,2 , S. Borin 1 , A. Rizzi 1 , H. Ouzari 2 , A. Boudabous 2 and D. Daffonchio 1 1 Dipartimento di Scienze e Tecnologie Alimentari e Microbiologiche, Universita` degli Studi, Milano, Italy and 2 Laboratoire de Microbiologie, Campus Universitaire, Universite´ de Tunis, Tunis, Tunisia 2002 ⁄ 10: received 15 January 2002, revised 29 April 2002 and accepted 13 May 2002 A. CHERIF, S. BORIN, A. RIZZI, H. OUZARI, A. BOUDABOUS AND D. DAFFONCHIO. 2002. Aims: To identify a chromosomal marker with signature nucleotides specific for Bacillus anthracis. Methods and Results: Repetitive element polymorphism-polymerase chain reaction with BOX-A1R primer was used to discriminate 52 strains of all six species of the ÔB. cereus groupÕ. A B. anthracis signature fragment, named AC-390, was cloned and sequenced. The deduced amino acid sequence was homologous to that of YwfK of B. subtilis. Using two internal primers, the AC-390 fragment was sequenced from two other B. anthracis strains as well as from strains of B. cereus and B. thuringiensis which have an AC-390 fragment homologous to that of B. anthracis as shown by Southern hybridization experiments. Conclusions: Two new signature sequences specific for B. anthracis were identified on a chromosomal fragment homologous to YwfK, a transcriptional regulator of B. subtilis. Significance and Impact of the Study: These results show a new chromosomal DNA trait useful for distinguishing B. anthracis from the related species of the B. cereus group, regardless of the presence of the virulence plasmids pXO1 and pXO2. INTRODUCTION Bacillus anthracis, a pathogen found throughout the world, causes anthrax in mammals (Mock and Fouet 2001). Outbreaks occur not only in wild animals (Smith et al. 1999) but also in domestic livestock, representing a severe economic threat (Patra et al. 1998). There is also great concern about the potential use of natural or engineered strains as biological weapons (Jackson et al. 1998; Turnbull 1999) and recent dramatic events have shown that this bacterium can be used in bio-terrorist attacks. Several authors have raised the question of the need for systems for the definitive identification of B. anthracis strains (Daffonchio et al. 1999; Ramisse et al. 1999; Turnbull 1999). Until now, the polymerase chain reaction (PCR)- based identification of B. anthracis has been based mainly on plasmidic genes (see, for example, Makino et al. 1993; Reif et al. 1994; Cheun et al. 2001) but it has been clearly highlighted that there is a need for molecular tools for a plasmid-independent, definitive identification of B. anthracis (Turnbull 1999). Very few chromosomal elements that can be used to discriminate B. anthracis from the closely related species of the ÔB. cereus groupÕ have been identified; they include the Ba813 fragment (Patra et al. 1996; Ramisse et al. 1996), the vrrA variable number tandem repeat (VNTR) (Andersen et al. 1996), other VNTR fragments used in a multiple-locus VNTR analysis (Keim et al. 2000), the SG-749 fragment (Daffonchio et al. 1999) and the rpoB gene (Qi et al. 2001). The wide application of published primers has brought to light some of the limitations of most of these primer sets, targeting plasmids (Beyer et al. 1999) as well as chromosomal DNA traits (Patra et al. 1998; Ramisse et al. 1999). For example, the Real-Time PCR assay on the rpoB gene developed by Qi et al. (2001) cross-reacted with a non-B. anthracis strain. These observations have revealed the need to search for new chromosomal traits that could act as additional and, perhaps, alternative tools to present-day chromosomal markers. Correspondence to: D. Daffonchio, Dipartimento di Scienze e Tecnologie Alimentari e Microbiologiche, Universita`degli Studi di Milano, via Celoria 2, 20133, Milano, Italy (e-mail: daniele.daffonchio@unimi.it). ª 2002 The Society for Applied Microbiology Journal of Applied Microbiology 2002, 93, 456–462