Strategy for Identification of Bacillus cereus and Bacillus thuringiensis ...

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 2006, p. 1295–1301 0099-2240/06/$08.00⫹0 doi:10.1128/AEM.72.2.1295–1301.2006 Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Vol. 72, No. 2

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, Universita ` Degli Studi di Milano, 20133 Milan, Italy1; G. Eliava Institute of Bacteriophage, Microbiology and Virology, 0160 Tbilisi, Georgia2; Laboratoire de Microbiologie, De´partement de Biologie, Faculte´ des Sciences de Tunis, 2092 Tunis, Tunisia3; Istituto Nazionale per le Malattie Infettive I.R.C.C.S. ‘Lazzaro Spallanzani,’ 00142 Rome, Italy4; and 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. anthracis that were absent in these closely related strains (34). In population genetics studies among a strain collection of the B. cereus group species, it was found by multilocus enzyme electrophoresis (23) and multilocus sequence typing (MLST) (24) that the strains could be divided into two main groups, the first including soil and dairy isolates and the second including strains with pathogenic potential. This second group included, besides B. anthracis, most of the strains isolated from patients in clinical environments and, among these, B. cereus strains isolated from periodontitis in humans. Helgason et al. (22, 24) clearly showed that in the species B. cereus and B. thuringiensis some genetic types exist having a close relationship with B. anthracis. Very recently, a human isolate that had been identified as B. cereus on the basis of phenotypic and molecular data caused clinical symptoms similar to those of inhalation anthrax. It was shown to harbor a virulence plasmid very similar to pXO1, together with a capsule plasmid was completely different than pXO2 (25). Considering all of these data and the potential virulence shown by strains that are genetically similar to B. anthracis, approaches that allow the rapid identification of such strains are great interest to gain further insight into B. anthracis virulence mechanisms, as well as to prevent the use of such strains for B. anthracis-based bioweapon development. The most common strategies to analyze the genetic relationship between B. anthracis, B. cereus, and B. thuringiensis are based on genome fingerprinting, e.g., AFLP (29, 34, 40) or repetitive element polymorphism-PCR (rep-PCR) (11). Alternatively, the analysis of conserved molecular chronometers

Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis are rather important microorganisms that interfere with or are related to human activities. B. anthracis is the active agent of anthrax disease (41), B. cereus causes food-borne disease syndromes associated with enterotoxin and emetic toxin (17, 27), and B. thuringiensis is an insect pathogen (39) currently used for the biological control of insects in crop protection. On the basis of genetic evidence, it has been proposed that B. anthracis, B. cereus, and B. thuringiensis belong to the same species (B. cereus sensu lato), but the status of separate species has been retained due to the remarkably different virulence phenotype (23). It has been hypothesized that B. anthracis derives from B. cereus-B. thuringiensis by the acquisition of the virulence pXO plasmids (29) and successive genome adaptation (30). It is thus predictable that B. cereus-B. thuringiensis strains exist that are genetically and phylogenetically closely related to B. anthracis. Several studies have shown that certain B. cereus strains are strictly related to B. anthracis. By means of amplified fragment length polymorphism (AFLP) typing, Radnedge et al. (34) individuated B. cereus and B. thuringiensis strains closely related to B. anthracis, and then tried to identify by suppression subtractive hybridization genomic regions of B.

* Corresponding author. Mailing address: Dipartimento di Scienze e Tecnologie Alimentari e Microbiologiche, Universita` degli Studi di Milano, via Celoria 2, 20133 Milan, Italy. Phone: 39-0250316730. Fax: 39-0250316694. E-mail: [email protected]. † Supplemental material for this article may be found at http://aem .asm.org/. 1295

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TABLE 1. Strains of the six species of the B. cereus group used in this study and RSI-PCR haplotypes determined by analyzing specific mutations of the long ITS containing tRNA genes RSI-PCR haplotype (no. of strains)

Species a (no. of strains)

H1 (21)

BA (18)

H2 (58)

BC (2) BT (1) BC (23) BM (2) BT (33)

H3 (33)

H4 (10) H5 H6 H7 H8

(1) (1) (1) (1)

BC (11) BM (10) BP (6) BT (6) BM (4) BT (2) BW (4) BT (1) BM (1) BM (1) BC (1)

Primer set c

Strain(s) b

170, 227, 300, 376, 256, 282, 582, 663, 779, 832, 846, 957, 4229, 6602, 7700, 7702, Cepanzo, Davis TE702 DSMZ 318, DSMZ 336 Sam2 DSMZ 31T, DSMZ 345, DSMZ 360, DSMZ 487, DSMZ 626, DSMZ 2896, Bc2, Cer1, Cer3, Cer5, Co1, Co2, My1, IO200, Bx1, Bx8, Bx11, Bx12, Bx19, Bx20, Bx21, Bx22, Bg7 NRS273T, A81 DSMZ 2046T subsp. thuringiensis, 1T subsp. thuringiensis, HD2 subsp. thuringiensis, DSMZ 5724 subsp. israelensis, 14T subsp. israelensis, DSMZ 5725 subsp. kurstaki, 33T subsp. kurstaki, HD1 subsp. kurstaki, BTK subsp. kurstaki, BTX subsp. kurstaki, PTB subsp. kurstaki, Bx16 subsp. kurstaki, 44T subsp. dendrolimus, 55T subsp. galleriae, 13T subsp. pakistani, 9T subsp. tolworthi, BMG1.1, BMG1.2, BMG1.7, BMG1.8, BMG1.9, BST21, BST22, BST23, BST25, BST26, BST30, Bac16, Bac24, Bac31, BacSH9, Bx3, Bx9 DSMZ 6127, DSMZ 46321, Bc1, Cer4, Cer6, MyD, Po1, AH812, AH817, AH818, AH831 DSMZ 303, DSMZ 307, Bif, G1, MycH, Nov1, Nov2, NRS 306, NRS 319, TP1 A82, B618, B346, BD10, CA, Tp2 7T subsp. aizawai, BMG1.3, BMG1.4, BMG1.5, BST33, Sam3 DSMZ 2048T, DSMZ 299, DSMZ 309, BmF HD868 subsp. tochigiensis, BMG1.6 10204T, 10201, 10202, 10208 10T subsp. darmstadiensis Bms B615 DSMZ 351

A

B

C

D

AB

⫺

⫺

⫾

⫹

⫺/⫺

⫹

⫹

⫺

⫺

⫹/⫹

⫹

⫺

⫺

⫺

⫹/⫺

⫺

⫺

⫺

⫺

⫺/⫺

⫺ ⫾ ⫹ ⫺

⫹ ⫺ ⫾ ⫹

⫺ ⫺ ⫺ ⫺

⫺ ⫺ ⫺ ⫺

⫺/⫹ ⫾/⫺ ⫹/⫾ ⫺/⫹

a

BA, B. anthracis; BC, B. cereus; BM, B. mycoides; BP, B. pseudomycoides; BT, B. thuringiensis; BW, B. weienstephanensis. For strain characteristics, please refer to tables published in references 11 and 16. c The nucleotide positions of the “cut primer” 3⬘ end in the sequence of the long ITS-containing the tRNA gene (accession no. AJ420048) of B. anthracis strain 7700 (10) and the restriction enzymes used were: primer set A, 75 and EcoRI; primer set B, 122 and PstI; primer set C, 122 and EcoRI; primer set D, 120 and NruI; and primer set AB, 75 and 120 and EcoRI and PstI, respectively. A “⫹” symbol indicates that the RSI-PCR product has been cut by the restriction enzyme used, while a “⫺” symbol indicates that the cut did not occur. A “⫾” symbol indicates that a partial restriction occurred in repeated experiments, indicating an interoperonic polymorphism in the target nucleotide position. b

could be used. The most used locus to describe genetic relationship and phylogeny among organisms is the ribosomal operon, especially the 16S and 23S rRNA genes (4, 5). However, these genes are much conserved among B. anthracis, B. cereus, and B. thuringiensis, and they cannot be used to unambiguously discriminate at the species level (6). With respect to the ribosomal genes, the intergenic transcribed spacers (ITS) between the 16S and 23S rRNA genes are hypervariable and can display polymorphisms especially in regions not implicated in rRNA maturation (19, 20). We previously showed that the ITS containing tRNA shows several single nucleotide polymorphisms (SNP) specific for B. anthracis (10). In light of the relative divergence of ITS, the finding of B. anthracis-specific SNP in B. cereus strains could indicate of a strict genetic relationship with B. anthracis. To test this hypothesis, we used restriction site insertionPCR (RSI-PCR) (13), a method designed to identify specific SNP, to screen a collection of 126 strains of the B. cereus group, over two previously discovered B. anthracis-specific SNP in the tRNA gene containing ITS (10). Two B. cereus and one B. thuringiensis strain with the same SNP pattern as B. anthracis were identified. MLST (24, 32) was thus used to confirm these strains as “near neighbors” of B. anthracis, together with four strains previously isolated from patients affected by periodon-

titis and thus far recognized as the strains closest to B. anthracis (22, 24). MATERIALS AND METHODS Bacterial strains and DNA extraction. The strains used in the present study (Table 1) (11, 16) were grown routinely as previously reported (7, 12, 15, 16), and DNA suitable for amplification was obtained by sodium dodecyl sulfate-proteinase K treatment (38) as already described (3, 12, 15, 16). RSI-PCR screening. For RSI-PCR, the following five primer sets were used: set A, ECO-CER-RSI-F (5⬘-TCGTTTAGTTTTGAGAGTTGAAT-3⬘) and ITSB-r (5⬘-GTGGGTTTCCCCATTCGG-3⬘); set B, ITS-A-f (5⬘-CCTTGTACA CACCGCCCGT-3⬘) and PST-CER-RSI-R (5⬘-GAGCTATAGGCCCCATAC AAACT-3⬘); set C, ITS-A-f (5⬘-CCTTGTACACACCGCCCGT-3⬘) and ECOANT-RSI-R (5⬘-GCTGAGCTATAGGCCCATACAGAAT-3⬘); set D, NRUANT-RSI-F (5⬘-GATATGATATAAATAAATCGCG-3⬘) and ITS-B-r (5⬘-GTG GGTTTCCCCATTCGG-3⬘); and set AB, a combination of previously mentioned primers ECO-CER-RSI-F and PST-CER-RSI-R. For all of the primer sets, initial denaturation was at 94°C for 4 min, followed by 35 cycles consisting of 94°C for 30 s, annealing phase specific for each primer set, and fragment extension at 72°C for 45 s, and a final extension step at 72°C for 10 min. The annealing conditions were 53°C for 45 s (set A), 57°C for 45 s (set B), 57°C for 45 s (set C), 50°C for 45 s (set D), and 53°C for 45 s (set AB). PCRs were carried out in 50-␮l mixtures in an I-cycler (Bio-Rad, Milan, Italy) with 1.25 U of Taq DNA polymerase (Amersham Pharmacia Biotech, Milan, Italy), 1⫻ Mg-free PCR buffer, each deoxynucleoside triphosphate at a concentration of 200 ␮M, 2.5 mM MgCl2, each primer at a concentration of 0.3 ␮M, and 2 ␮l of bacterial DNA. PCR products were digested overnight with the respective restriction enzyme(s) (Table 2) and separated by standard agarose gel electrophoresis (38).

B. CEREUS GROUP STRAINS RELATED TO B. ANTHRACIS

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TABLE 2. Comparison of “cut primer” sequence and target sequences in B. anthracis and B. cereus a Primer set b

SNP positionc

A B C D AB

75 121 121 121 75 and 121

Primer sequence upstream the SNP 5⬘- . 5⬘- . 5⬘- . 5⬘- . Like

. . gagttgaat-3⬘ . . tatgtttga-3⬘ . . tatgtctta-3⬘ . . taaatcgcg-3⬘ primer sets A and B

Target sequence in B. anthracis d

Target sequence in B. cereuse

Restriction site(s) created by RSI-PCR (restriction enzyme)

Target species f

gagttcaatAAaaagt atcctgAaatttgtat cctgAaatttgtatgg aaataaatcctgAaat Like primer sets A and B

gagttcaatTCa––gt atcctgCaatttgtat cctgCaatttgtatgg aaataaatcctgCaat Like primer sets A and B

GAATTC (EcoRI) CTGCAG (PstI) GAATTC (EcoRI) TCGCGA (NruI) Like primer sets A and B

BC/BT BC/BT BA BA BC/BT

a The cut primer is defined as the primer that inserts a restriction site in the target sequence during PCR and is eliminated by the following restriction digestion (13). Underlined in the primer and the target sequences are the nucleotides modified in the primer to insert the appropriate restriction site with respect to a specific SNP in the target sequence. In the target sequences, species-specific SNP are marked in boldface uppercase letters. b Primer sets referred to Fig. 2. For the complete primer sequence, see Materials and Methods. c SNP positions based on the sequence coordinates of B. anthracis 7700 (accession no. AJ420048) (10). d Nucleotide sequence based on the sequence of B. anthracis 7700 (accession no. AJ420048) (10). e Nucleotide sequence based on the sequence of B. cereus 31T (accession no. AJ420052) (10). f BA, B. anthracis; BC, B. cereus; BT, B. thuringiensis.

MLST. Five loci were used for MLST: the 16S-23S rRNA long ITS, which contains tRNA genes (10); the SG-749 fragment that includes a region homologous to the B. subtilis ypuA gene (14); the AC-390 fragment that is homologous to the B. subtilis ywfK gene, encoding a hypothetical transcriptional regulator belonging to the LysR family (9); the plcR gene encoding for a pleiotropic regulator previously identified as one of the principal regulators of B. cereus virulence genes (1); and the cerA gene that encodes the cereolysin A phospholipase (18). For sequencing the long 16S-23S rRNA ITS containing tRNA genes, a previously described procedure (10) was followed. The other four loci were amplified and sequenced by using the following primers: YWFK-F (5⬘-GAAAATGGC CGGATGAGT-3⬘) and YWFK-R (5⬘-GACGTTGAAACATTTATGCA-3⬘) for AC-390; PLCR-F (5⬘-GAAGTGAAATTAAGAAAATTAG-3⬘) and PLCR-R (5⬘-TATAATGCTTTTGCATGATTAT-3⬘) for plcR; CERA-F (5⬘-GAGTTT AGAGAACGGTATTTATGCTGC-3⬘) and CERA-R (5⬘-CTACTGCCGCTC CATGAATCC-3⬘) for cerA; and SG749-F (5⬘-ACTGGCTATTATGTAATG-3⬘) and SG749-R (5⬘-ATAATTATCCATTGATTT-3⬘) for SG-749. DNA fragments were sequenced on both strands with the DYEnamic ET terminator cycle sequencing kit (Amersham Pharmacia Biotech) with the primers used to generate the PCR products in an ABI Prism 310 DNA capillary sequencer (Applied Biosystems, Monza, Italy). Each sequence was checked manually and searched for sequence similarities in databases with the assistance of the BLAST facilities (2). Data analysis. Phylogenetic clustering of the strains was done with CLUSTAL W alignment tools and the TREECON 1.3b package (42) and was based on the total numbers of differences among gene sequences performed by using the neighbor-joining method. Bootstrap analysis was used by resampling the sequence alignment 1,000 times. In MLST analysis the strains of the “B. cereus group” with complete genome sequences available in GenBank were used as reference strains. The strains and their accession numbers are as follows: B. anthracis Ames NC003997, B. anthracis Ames Ancestor NC007530, B. anthracis Sterne NC005945, B. cereus ATCC 10987 NC003909, B. cereus ZK NC006274, B. cereus ATCC 14579 NC004722, and B. thuringiensis serovar Konkukian NC005957. Nucleotide sequence accession numbers. Nucleotide sequences have been deposited in the GenBank data bank under accession numbers AM062640 to AM062674.

RESULTS AND DISCUSSION Primer selection for SNP detection in the 16S-23S rRNA ITS by RSI-PCR. Several methods based on whole genome fingerprinting, such as MLST (24), AFLP (29), or rep-PCR (11) have been used to differentiate near neighbors of B. anthracis. However, signature SNP in housekeeping genes could also be exploited to identify strains related to certain species or genetic types. For example, Pruss et al. (33) showed that T and A nucleotides in certain positions in the 16S rRNA gene, and their prevalence among the different ribosomal operons correlate with the psychrotolerance within the B. cereus group and represent signatures of the species B. weihenstephanensis.

We focused on a region of the ribosomal operon, the long ITS containing tRNA genes, by evaluating whether signature nucleotides that have been shown to be specific for B. anthracis (10) would be shared by B. cereus and B. thuringiensis strains, thereby reflecting a widespread similarity of their genome with that of B. anthracis. The rationale behind the choice of the ITS is that this region is less conserved in the ribosomal operon than the adjacent structural rRNA genes. However, the ITS regions are involved in the maturation process of rRNAs by forming double-stranded stretches with the region upstream the 16S rRNA gene. RNase III that cleaves the primary rRNA transcript, starting the maturation process, recognizes these double-stranded regions. This mechanism explains the conservative nature of the ITS shown by the absence of speciesspecific signature in the short ITS of the B. cereus group species (8, 10, 21). The presence of tRNA genes, however, determines in the primary rRNA transcript the formation of singlestranded bending regions around the tRNA secondary structure that are probably not involved in the rRNA maturation process. These nucleotides could be more prone to mutations between species or bacteria colonizing specialized environmental niches such as B. anthracis (28). To test the hypothesis that B. anthracis-specific SNP of the 16S-23S rRNA ITS could be useful for the identification of near neighbors B. cereus and B. thuringiensis, we chose two nucleotide positions (75 and 121) upstream of the first tRNA gene (tRNAIle) of the long ITS of B. anthracis 7700 (accession no. AJ420048) (10) (Fig. 1a). In a recent study aimed to develop a microarray-based tool for the rapid identification of B. anthracis, position 121 has been confirmed to be B. anthracis specific, whereas position 75 cross-reacted with two B. cereus strains (ATCC 10987 and G9241) (31). We have selected five primer sets, one specifically targeting position 75, three for position 121, and one simultaneously targeting both positions (Table 2). The criteria used for the primer design were those reported by Daffonchio et al. (13), i.e., leaving the 3⬘ end nucleotide unmodified with respect to the target sequence and making a minimal number of primer-template mismatches to insert restriction sites for six base-pair-recognizing restriction enzymes that do not cut the original target sequence. Primer set AB was designed to simultaneously detect both of the SNP using the appropriate restriction enzyme.

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FIG. 1. (a) Scheme of the RSI-PCRs designed to study a collection of B. cereus group strains for the presence of two SNP previously identified as species-specific for B. anthracis (10). The two SNP identified by triangles were located at positions 75 and 121, as shown in Table 1. A to D indicate the relative schematic positions of the four primer sets and related RSI-PCR fragments in the map of the ribosomal spacer containing tRNA genes. In the A and D primer sets the cut primer corresponded to the forward primer, while in sets B and C it was designed as a reverse primer. The normal primers were designed on the 16S rRNA gene (sets B and C), the 23S rRNA gene (sets A and D) or they were internal to the ITS (set AB). (b to e) Examples of the results of the RSI-PCR screening of two specific nucleotides in the ITS containing tRNA genes in the six species of the B. cereus group. Lanes M, 50-bp ladder. The arrow marks the position of the 250-bp fragment. (b) RSI-PCR with primer set D and endonuclease NruI. Lanes: 1 and 2, B. anthracis 7700 after and before restriction digestion; 3 and 4, B. anthracis 282 after and before restriction digestion. (c) RSI-PCR with primer set D and endonuclease NruI. Lane: 1, B. mycoides Bif; 2 to 4, B. cereus DSMZ 360, DSMZ 336, and DSMZ 318; 5 and 6, B. anthracis Cepanzo and 376; 7 to 9, RSI-PCR products before restriction digestion of B. cereus DSMZ 336 and DSMZ 318 and B. anthracis 376. (d) RSI-PCR with primer set AB and restriction digestion with endonucleases EcoRI and PstI. Lanes: 1 to 6, B. anthracis 957, Cepanzo, 6602, 4229, 256, and 300; 7 to 10, B. thuringiensis BST23, BMG1.4, BMG1.5, and BMG1.6; 11 to 18, B. cereus Cer1, Bc2, Bx12, Bx19, Co2, Bx1, DSMZ 318, and DSMZ 360; 19, undigested RSI-PCR product of B. cereus Cer1. (e) RSI-PCR with primer set C and restriction digestion with endonuclease EcoRI. Lanes: 1 to 3, B. anthracis Davis TE702, 957 and Cepanzo; 4, undigested RSI-PCR product of B. anthracis 4229.

The five primer sets were designed to have different combinations of the “cut primer” (13) and the reverse primer and to target B. anthracis or B. cereus-B. thuringiensis specific SNP (Table 2 and Fig. 1a). In primer set A, the cut primer targeted position 75 of the ITS by inserting an EcoRI site in B. cereus-B. thuringiensis, while the reverse primer was a universal primer on the 5⬘ end of the 23S rRNA gene. The cut primers in primer sets B and C were designed to target position 121 of the ITS of B. cereus-B. thuringiensis and B. anthracis, respectively, by introducing two different restriction sites after amplification. These two primers were coupled with two different forward primers designed on conserved regions of the ITS and the 16S rRNA gene. The cut primer of primer set D targeted nucleotide 121 of the ITS by inserting an NruI site specific for B. anthracis. It was coupled with a reverse primer targeting a

universal sequence stretch of 23S rRNA gene. In primer set AB the cut primers of sets A and B were used in combination as forward and reverse primers. This primer set was designed to simultaneously detect in a single RSI-PCR test two nucleotides at positions 75 and 121 specific for B. cereus and B. thuringiensis. RSI-PCR screening of B. cereus group strains. A total of 126 strains of the six species of B. cereus group were analyzed for the presence of two specific nucleotides by using five RSI-PCR primer sets (Tables 1 and 2). Examples of the agarose gel electrophoresis profiles are shown in Fig. 1. By comparing RSI-PCR products in agarose gel electrophoresis before and after restriction digestion, it was possible to appreciate the elimination of the cut primer by the endonuclease digestion when the suitable restriction site was inserted by the PCR (Fig.

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FIG. 2. Phylogenetic relationship among B. anthracis (Ba), B. cereus (Bc), and B. thuringiensis (Bt) strains as determined by MLST. The presented strains were identified as near neighbors of B. anthracis by RSI-PCR on species-specific SNP in the long ITS containing tRNA genes. The tree was determined by neighbor-joining analysis of about 2,300 nucleotide positions from the sequencing of five independent genetic loci, the 16S-23S rRNA ITS containing tRNA, plcR, SG-749, AC-390, and cerA. The bar indicates 1% of phylogenetic distance. Bootstrap values are indicated.

1b to d). Primer set AB enabled detection of one or, after the cut of both the cut primers, two SNP in a single reaction (Fig. 1d). In several strains, after endonuclease digestion with some primer sets, two bands were detected, one corresponding to the undigested RSI-PCR product and the other to the product after the elimination of the cut primer. This was the case, for example, of B. anthracis strains, two B. cereus strains and one B. thuringiensis strain, when analyzed for a SNP at position 121 of the ITS with primer set C and the restriction endonuclease EcoRI (Table 1 and Fig. 1e). To evaluate whether these double bands were due to partial endonuclease digestions, the experiments were repeated several times, always giving the same results. This indicates that interoperonic polymorphisms characterize nucleotide position 121 of the ITS in these strains. The results of the RSI-PCR screening for the whole strain collection are reported in Table 1. By putting together the data obtained with all of the primer sets, eight RSI-PCR haplotypes could be detected, named H1 to H8. This result confirmed the relatively high polymorphism in the B. cereus group. In contrast, B. anthracis was confirmed to be highly monomorphic, all of the strains being grouped in a single haplotype (H1). About

62 and 77% of the B. cereus and B. thuringiensis strains, respectively, were grouped in RSI-PCR haplotype H2. Most of the remaining B. cereus (30%) and B. thuringiensis (14%) strains were grouped in RSI-PCR haplotype H3, which also included 55% of B. mycoides and all B. pseudomycoides strains. All of the B. weihenstephanensis and 22% of the B. mycoides strains were grouped in RSI-PCR haplotype H4. Although B. cereus and B. thuringiensis were confirmed to be polymorphic species, being distributed in several haplotypes, their relative distributions among the different groups indicated that one haplotype (H2) accounts for a high percentage of strains and could therefore represent a major lineage in the two species. Interestingly, two B. cereus strains (DSMZ 318 and DSMZ 336) and one B. thuringiensis strain (Sam2) were grouped with B. anthracis strains in RSI-PCR haplotype H1, indicating that they shared the same SNP at position 75 and 121 of the ITS. Remarkably, the strain collection used in the present study included unrelated randomly chosen isolates, except for the B. cereus clinical strains associated with periodontitis (AH812, AH817, AH818, and AH831), which were previously shown to be genetically related to B. anthracis. By considering the 76

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randomly chosen B. cereus-B. thuringiensis (excluding the periodontitis isolates), the three strains identified as B. anthracis near neighbors by RSI-PCR represent ca. 4% of the B. cereus-B. thuringiensis strains examined. Although this percentage should be confirmed by the analysis of a larger collection of isolates from different origin and geographical locations, it suggests that a minority of B. cereus-B. thuringiensis strains is strictly related to B. anthracis. This reinforces the hypothesis that B. anthracis is only recently derived from a specific genotype of B. cereus (29, 30). MLST of B. cereus group strains as potential near neighbors of B. anthracis. Based on the similarity of the B. anthracis RSI-PCR patterns, B. cereus DSMZ 318 and DSMZ 336 and B. thuringiensis Sam2 were considered as potential near neighbors of B. anthracis and were further investigated by MLST analysis. These strains were compared to several strains from the “B. cereus group,” such as B. anthracis Ames, B. anthracis Ames Ancestor, B. anthracis Sterne, B. thuringiensis serovar Konkukian, and B. cereus ATCC 10987, ATCC 14579, and ZK and to several B. cereus strains (AH812, AH817, AH818, and AH831) previously indicated among the closest strains to B. anthracis on the basis of multilocus enzyme electrophoresis (22, 23) and MLST (24). The phylogenetic tree, inferred from the comparison of about 2,300 nucleotide positions from five genetic loci, is reported in Fig. 2. The tree shows three B. cereus strains—DSMZ 336, DSMZ 318, and AH818—to be the most closely related to B. anthracis, followed by B. thuringiensis serovar Konkukian, B. cereus ZK, and B. thuringiensis Sam2. B. cereus ATCC 10987 and the other three B. cereus periodontitis isolates AH812, AH817, and AH831 were more distantly related. B. cereus ATCC 14579 resulted the most distant from B. anthracis, confirming what was already observed by MLST (24, 25) and genome sequencing (26). By separately analyzing the phylogenetic relationship among the strains at each locus (Fig. S1 in the supplemental material), it was found that B. cereus strains DSMZ 336 and DSMZ 318 were the most closely related to B. anthracis at housekeeping loci such as the long ITS containing tRNA genes and AC-390, in the case of the latter, along with strain Sam2. B. cereus AH818 was the closest to B. anthracis at locus SG-749. B. cereus DSMZ 336 was the strain closest to B. anthracis in the plcR sequence, whereas strain AH818 was the closest to B. anthracis in the cerA sequence. The periodontitis strains AH812 and AH831 were grouped together for all of the sequences examined, while strain AH817 was segregated based on the long ITS-containing tRNA gene sequence, in agreement with another MLST scheme (24) and a comparative genome hybridization study (37). According to the same previous MLST scheme, B. cereus ATCC 10987 (36) was confirmed here to diverge from B. anthracis, analogously to strains AH812, AH817, and AH831, indicating that it is not among the strains most closely related to B. anthracis. The strains closely related to B. anthracis—DSMZ 336, DSMZ 318, AH818, and Sam2, as well as strains AH812, AH817, and AH831—were tested for the presence of B. anthracis virulence genes cap, pag, lef, and cya by PCR using the primer sets described by Ramisse et al. (35), but none of them were positive for any of the four genes (data not shown). These strains also appeared to lack plasmids, as detectable by


agarose gel electrophoresis of total DNA from bacterial clear lysates (data not shown) (15). By aligning the sequences of B. anthracis and the nearneighbor B. cereus and B. thuringiensis strains DSMZ 336, DSMZ 318, AH818, and Sam2 and the B. cereus strains AH812, AH817, and AH831 obtained for the five loci used in the MLST, signature nucleotides in SG-749, plcR, and cerA genes were identified, offering the opportunity for designing species-specific probes or primers for the rapid identification of B. anthracis, e.g., by RSI-PCR (13). By referring to the sequence coordinates of the three loci SG-749, plcR, and cerA (accession no. NC003997), B. anthracis can be distinguished from near neighbors as follows (B. anthracis versus B. cereus and B. thuringiensis): SG-749 at positions 71 (T versus C), 122 (G versus A), 143 (C versus T), 538 (A versus C), and 581 (C versus G); plcR at position 361 (C versus G); and cerA at positions 305 (C versus T). In conclusion, we showed here that SNP in conserved regions such as the ITS-containing tRNA genes can be useful signatures for the identification of B. cereus and B. thuringiensis strains that are “near neighbors” of B. anthracis. RSI-PCR resulted in a simple and efficient tool for tracing these SNP in large collection of strains, without requiring expensive equipment such as capillary electrophoresis systems. The availability of a wide collection of near-neighbor strains of B. anthracis would be a useful tool for the understanding of the evolution of this species within the B. cereus group, as well as for the detection of unambiguous DNA signatures for B. anthracis that can be used as markers for the detection of the pathogen. ACKNOWLEDGMENTS This study was supported in part by INTAS (International Association for the Promotion of Cooperation with Scientists from the New Independent States of the former Soviet Union) within the project “An Epidemiological Study of Outbreaks of B. anthracis in Georgia” (INTAS-01-0725) and by ISPELS within the project B74/MDL/02 to D.D. and P.V. M.M. was partially supported by a grant of the Italian Embassy in Tbilisi. A.C. was supported by a grant from the Direction Generale de Recherche Scientifique et Technologique of the Ministere de l’Education Superieure of Tunisia. We thank Miche´le Mock, Guy Patra, Samir Jaoua, Hala KhyamiHorani, Lawrence K. Nakamura, Siegfried Scherer, Ralf Mayr, and Daniel R. Zeigler for kindly giving us Bacillus strains and/or DNA. REFERENCES 1. Agaisse, H., M. Gominet, O. A. Okstad, A.-B. Kolsto, and D. Lereclus. 1999. PlcR is a pleiotropic regulator of extracellular virulence factor gene expression in Bacillus thuringiensis. Mol. Microbiol. 32:1043–1053. 2. Altschul, S. F., W. Gish, W. Miller, E. W. Myers, and D. J. Lipman. 1990. Basic local alignment search tool. J. Mol. Biol. 215:403–410. 3. Andreoni, V., M. Moro Luischi, L. Cavalca, D. Erba, and S. Ciappellano. 2000. Selenite tolerance and accumulation in the Lactobacillus species. Ann. Microbiol. 50:77–88. 4. Ash, C., and M. D. Collins. 1992. Comparative analysis of 23S rRNA gene sequences of Bacillus anthracis and emetic Bacillus cereus determined by PCR-direct sequencing. FEMS Microbiol. Lett. 94:75–80. 5. Ash, C., J. A. E. Farrow, S. Wallbanks, and M. D. Collins. 1991. Phylogenetic heterogeneity of the genus Bacillus revealed by comparative analysis of small-subunit-rRNA sequences. Lett. Appl. Microbiol. 13:202–206. 6. 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. 7. Borin, S., D. Daffonchio, and C. Sorlini. 1997. Single strand conformation polymorphism analysis of PCR-tDNA fingerprinting to address the identification of Bacillus species. FEMS Microbiol. Lett. 157:87–93. 8. Bourque, S. N., J. R. Vale´ro, M. C. Lavoie, and R. C. Levesque. 1995. Comparative analysis of the 16S to 23S ribosomal intergenic spacer se-

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