PCR Assay of the groEL Gene for Detection and Differentiation of Bacillus cereus Group Cells

Yu-Hsiu Chang, Yung-Hui Shangkuan, Hung-Chi Lin and Hwan-Wun Liu Appl. Environ. Microbiol. 2003, 69(8):4502. DOI: 10.1128/AEM.69.8.4502-4510.2003.

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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 2003, p. 45024510 0099-2240/03/$08.000 DOI: 10.1128/AEM.69.8.45024510.2003 Copyright 2003, American Society for Microbiology. All Rights Reserved.

Vol. 69, No. 8

PCR Assay of the groEL Gene for Detection and Differentiation of Bacillus cereus Group Cells
Yu-Hsiu Chang, Yung-Hui Shangkuan,* Hung-Chi Lin, and Hwan-Wun Liu
Division of Bacteriology, Institute of Preventive Medicine, National Defense Medical Center, Sanhsia, Taipei, Taiwan 237, Republic of China
Received 13 November 2002/Accepted 8 May 2003

Strains of species in the Bacillus cereus group are potentially enterotoxic. Thus, the detection of all B. cereus group strains is important. As 16S ribosomal DNA sequence analysis cannot adequately differentiate species of the B. cereus group, we explored the potential of the groEL gene as a phylogenetic marker. A phylogenetic analysis of the groEL sequences of 78 B. cereus group strains revealed that the B. cereus group strains were split into two major clusters, one including six B. mycoides and one B. pseudomycoides (cluster II) and the other including two B. mycoides and the rest of the B. cereus group strains (cluster I). Cluster I was further differentiated into two subclusters, Ia and Ib. The sodA gene sequences of representative strains from different clusters were also compared. The phylogenetic tree constructed from the sodA sequences showed substantial similarity to the tree constructed from the groEL sequences. Based on the groEL sequences, a PCR assay for detection and identication of B. cereus group strains was developed. Subsequent restriction fragment length polymorphism (RFLP) analysis veried the PCR amplicons and the differentiation of the B. cereus group strains. RFLP with MboI was identical for all the B. cereus group strains analyzed, while RFLP with MfeI or PstI classied all B. cereus and B. thuringiensis strains into two groups. All cluster II B. mycoides and B. pseudomycoides strains could be discriminated from other B. cereus group bacteria by restriction analysis with TspRI.

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Bacillus cereus group bacteria are environmental organisms which encompass at least four species: Bacillus anthracis, Bacillus cereus, Bacillus thuringiensis, and Bacillus mycoides. B. anthracis causes anthrax disease (48). B. cereus causes foodborne disease (13). B. cereus is also a plant growth promoter and an animal probiotic (14, 37, 52). B. thuringiensis is an insect pathogen (2). B. mycoides has recently been recognized as a plant growth-promoting bacterium associated with conifer roots (39). Recently, a Bacillus pseudomycoides (33) and a psychrotolerant Bacillus weihenstephanensis (31) were described. The taxonomy of this group is controversial, and several researchers have suggested that these closely related species should all be grouped as members of B. cereus (26, 44). Helgason et al. (24) used multilocus enzyme electrophoresis and sequence analysis of nine chromosomal genes to show that B. anthracis should be considered a lineage of B. cereus. Carlson et al. (8) found that neither pulsed-eld gel electrophoresis nor multilocus enzyme electrophoresis analysis could distinguish between B. cereus and B. thuringiensis species. The only established difference between B. cereus and B. thuringiensis strains is the presence of genes coding for the insecticidal toxins, usually present on plasmids. If these plasmids are lost, B. thuringiensis can no longer be distinguished from B. cereus (24). B. cereus is known to produce an emetic toxin, which causes vomiting, and at least three different enterotoxins, which cause diarrhea (18). There have been some indications that toxins
* Corresponding author. Mailing address: Institute of Preventive Medicine, National Defense Medical Center, P.O. Box 90048-700, Sanhsia, Taipei, Taiwan 237, Republic of China. Phone: (886 2) 8177 7038. Fax: (886 2) 2673 3025. E-mail: yhshangtw@yahoo.com. 4502

are produced by B. thuringiensis and B. mycoides. Damgaard (12) observed diarrheal enterotoxin production by strains of B. thuringiensis isolated from commercial B. thuringiensis-based insecticides. Beattie and Williams (5) detected toxigenic strains of B. thuringiensis and B. mycoides in a cytotoxicity assay. Perani et al. (38) also found B. cereus-type enterotoxins in natural isolates of B. thuringiensis. B. cereus is known to spoil milk and other food products (30). Therefore, its presence in food processing plants should be minimized. Accurate diagnostic tools are thus required to ensure the hygienic quality of susceptible food items. Several selective plating methods have been described for detecting B. cereus (46). The selection is based, for instance, on the ability of B. cereus to grow in the presence of polymyxin B and its lecithinase reaction. These methods require up to 4 days to perform due to the need for conrmatory testing. Another approach for detecting B. cereus is to amplify a specic, unique DNA sequence of the organism. DNA sequence analysis is the most precise analytical tool for microbial identication, and PCR has now made the characterization of gene segments from a large number of isolates feasible. The nucleotide sequence contains more etiologically specic information about evolution than the traditionally used phenotypic traits and is precisely dened and relatively simple to determine. Two important properties of a nucleotide sequence to be used for bacterial identication are that (i) it must be universal in its distribution and (ii) it must contain sufcient sequence variation. The most commonly used molecule is 16S rRNA because it is generally accepted that its sequence can be used to distinguish genera and well-resolved species (10, 35). However, 16S rRNA is limited in its ability to differentiate the B. cereus group bacteria. The nucleotide sequences of the 16S

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rRNAs of the B. cereus group exhibited very high levels of sequence similarity (99%) that were consistent with the close relationships shown by previous DNA hybridization studies (3). Likewise, Ash and Collins (4) reported that the 23S rRNA gene sequences of B. anthracis and an emesis-causing B. cereus strain were almost identical. In this study, we examined the phylogenetic relationships of B. cereus group strains on the basis of the nucleotide sequences of their groEL and sodA genes. The groEL genes, which encode highly conserved housekeeping proteins that assist in proper protein folding (also known as molecular chaperonins), are ubiquitous in both prokaryotes and eukaryotes. Viale et al. (49) and Gupta (20) previously observed that evolutionary trees drawn from the protein sequences of these molecules in eubacteria demonstrated remarkable similarity to those derived from 16S rRNA genes. Furthermore, the groEL sequences were found to be useful for species identication and taxonomic classication of the genus Staphylococcus (17). The sodA gene encodes a manganese-dependent enzyme (manganese-dependent superoxide dismutase). The use of a single pair of degenerate primers designed from the sodA gene constitutes a valuable approach to the genotypic identication of streptococcal (41) and enterococcal (42) species. The proteins encoded by the groEL and sodA genes are essential for cell survival in bacteria, and horizontal transmission of these genes may be as rare as that of rRNA genes. Protein-coding genes have been reported to evolve much faster than rRNA genes (34); thus, phylogenetic analysis with the groEL and sodA sequences is expected to provide higher resolution than one with 16S rRNA sequences. In order to investigate the possible characterization of the B. cereus group bacteria through groEL gene sequencing, the sequence of a 533-bp portion of this gene from 78 reference strains and soil isolates of the B. cereus group was determined and further analyzed. On the basis of their sequences, these strains were categorized into different clusters. Further evidence is presented that B. cereus group bacteria can be clearly differentiated into similar clusters by the sequences of the other protein-coding gene employed in this study, sodA. Based on these sequencing and phylogenetic data, PCR and PCRrestriction fragment length polymorphism (RFLP) methods were developed to identify and differentiate B. cereus group cells.
MATERIALS AND METHODS Bacterial strains. A total of 78 B. cereus group strains, including 56 reference strains and 22 Taiwan B. cereus soil isolates (Institute of Preventive Medicine [IPM] B. cereus soil [BCS] strains 3, 5, 8, 14, 17, 18, 19, 20, 21, 23, 26, 27, 29, 30, 31, 33, 34, 36, 37, 39, 41, and 42), as shown in Table 1, were used for sequencing and assayed for groEL RFLP types. A total of 38 Taiwan B. cereus soil isolates, including an additional 16 Taiwan B. cereus soil isolates (IPM BCS 1, 4, 6, 9, 10, 11, 12, 13, 15, 16, 22, 24, 25, 28, 38, and 40), were assayed for both vrrA RFLP and groEL RFLP types (see Table 4). To determine the specicity of our PCR primers, 13 non-B. cereus group Bacillus strains (Table 2) were used. The various Bacillus species isolates were grown at 30C on nutrient agar (NA) medium (Difco) overnight and used for DNA extraction. All isolates used were from the laboratory stock collections at the Institute of Preventive Medicine, National Defense Medical Center, Taipei, Taiwan, Republic of China. PCR amplication of groEL gene and sodA gene. Crude DNA extracts for PCR amplication were made from isolated colonies with the Instagene matrix (BioRad) according to the manufacturers protocol. An anticipated 600 bp of groEL DNA was amplied by PCR with primer set H279 and H280 (Table 3) (16).

Primers d1 and d2 (Table 3) (40) were also used to amplify an internal fragment representing approximately 85% of the sodA gene of the bacterial strains. The PCR protocols were as described in references 16 and 40, respectively. Purication of PCR products, cloning, and transformation. The amplied PCR products were puried with the QIAquick PCR purication kit (Qiagen). When multiple bands were visualized on the gel, the band was cut out and the DNA was puried with the QIAquick gel extraction kit. Cloning was performed with the TA cloning vector pCRII (Invitrogen) as described in the manufacturers protocol. DNA sequencing. Plasmids with the correct PCR insert from transformed Escherichia coli were puried with the Wizard Miniprep kit (Promega). DNA sequencing was performed by the uorescence-based dideoxy chain termination method with the universal sp6 promoter primer and T7 promoter primer in an automated DNA sequencer (Applied Biosystems model 373A). Data analysis. Sequence analysis was performed with the entire cloned fragment, omitting the primer sequences used to amplify the two different genes. Multiple alignment of the partial gene sequences was carried out with the Pileup program of the Genetics Computer Group package (Wisconsin package, version 9.1). Phylogenetic analysis was performed with the PHYLIP program package, version 3.57 (15). For the groEL genes, the published groEL gene sequences of B. subtilis (GenBank accession no. M84965) and B. stearothermophilus (GenBank accession no. AB028452) were included for comparison; the sequence of B. stearotheromophilus groEL was used as the outgroup. For the sodA genes, the published sodA gene sequences of B. licheniformis (GenBank accession no. AJ002279) and B. stearothermphilus (GenBank accession no. M81188) were included for comparison; the sequence of B. stearotheromophilus sodA was used as the outgroup. The phylogenetic tree was constructed by the neighbor-joining method with the JukesCantor correction for multiple substitutions according to the one-parameter model (25). Bootstrapping was performed with 1,000 iterations. The phylogenetic tree was drawn with TreeView software. Specic PCR amplication of part of groEL gene and groEL RFLP analysis from B. cereus group. (i) PCR amplication of part of groEL gene. An internal portion of the groEL gene was amplied by PCR with our designed primer set (ba1F and ba1R; see Table 3). Then 4 l of InstaGene DNA extract was used as the source of DNA template in the PCR. The PCR mixture (50-l volume) contained 1.8 U of AmpliTaq DNA polymerase (Perkin Elmer), 10 mM Tris-HCl (pH 8.5), 50 mM KCl, 1.5 mM MgCl2, 0.01% (wt/vol) gelatin, 200 M each of the deoxynucleoside triphosphates, and 0.5 M each of two opposing primers. Amplication was performed in a thermal cycler (MJ PTC-100-60) with temperature ramping as follows: 94C for 2 min to denature the template, followed by 35 cycles of 94C for 1 min, 55C for 1 min, and 72C for 1 min, and nally 72C for 5 min. (ii) groEL RFLP analysis. For groEL RFLP analysis, 5 to 10 l of PCR product obtained with the ba1F and ba1R primers was combined with the appropriate restriction enzyme buffer and endonuclease as outlined by the manufacturer (New England Biolabs, Inc. Beverly, Mass.) and incubated at 37C for 1 to 3 h. groEL RFLP products were analyzed by agarose gel electrophoresis. vrrA RFLP analysis. vrrA PCR products were amplied with primers GPR6 and GPR7 (Table 3). vrrA RFLP was done by the method of Shangkuan et al. (45). Nucleotide sequence accession numbers. Representative nucleotide sequences of the internal fragment of the groEL genes have been submitted to the GenBank data bank and assigned the following accession numbers: AY112842 to AY112852 for reference B. cereus strains; AY112854 to AY112863 for reference B. thuringiensis strains; AY112864 to AY112869 for reference B. mycoides strains; AY1112870 for B. pseudomycoides DSM 12442; AY112871 for B. anthracis ATCC 11966; and AY112820 to AY112841 for Taiwan B. cereus soil isolates. Representative nucleotide sequences of the internal fragment of the sodA genes have been submitted to the GenBank data bank and assigned the following accession numbers: AY112885 to AY112888 and AY112890 to AY112893 for reference B. cereus strains; AY112876 to AY112884 for reference B. thuringiensis strains; AY112873 to AY112875 for reference B. mycoides strains; and AY112872 for B. anthracis ATCC 11966.

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RESULTS Cloned partial groEL gene sequences of B. cereus group strains. Sequences were determined for the groEL PCR products generated from 56 reference B. cereus group strains and 22 Taiwan B. cereus soil isolates. A sequence of at least 533 bp was

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TABLE 1. Fifty-six reference B. cereus group strains and 22 Taiwan B. cereus soil isolates sequenced and analyzed for groEL phylogenetic groups and assayed for groEL RFLP types
Groupa Strainsb Restriction digestion pattern no.c MboI MfeI PstI TspRI RFLP type groEL phylogenetic group

II III IV V VI VII

VIII IX X XI XII XIII XIV XV XVI XVII

B. anthracis ATCC 11966, 14186, 14187, 937, 4728, 6603, 9660, 10, 14578; NCTC 8234, CDC 024/66NH, IPM BA13; B. cereus ATCC 11950 B. cereus NCTC 11143, ATCC 13061, 11778 B. cereus ATCC 9818, 49064, 21282, 21769, IPM BCS 30, 33, 37, 42 B. thuringiensis CCRC 14373, 14375 B. cereus IPM BCS 5 B. cereus CCRC 10250, ATCC 31293 B. thuringiensis CCRC 11500, 15655, 11498, 13838, 13849, ATCC 33679, 35866; B. cereus ATCC 21281, 27348, IPM BCS 8, 21 B. cereus 43881, IPM BCS 17, 20, 27, 29; B. thuringiensis CCRC 11503 B. cereus ATCC 33019, 19637 B. cereus IPM BCS 14, 26; B. thuringiensis ATCC 10792, 13367, CCRC 15666 B. thuringiensis CCRC 14377; B. cereus ATCC 31429, IPM BCS 23, 34 B. thuringiensis CCRC 14374, 14376 B. cereus IPM BCS 18, 19 B. cereus ATCC 14579, IPM BCS 3, 31, 36, 39, 41; B. thuringiensis CCRC 11501 B. cereus ATCC 21182; B. mycoides ATCC 6462, CCRC 11967 B. mycoides ATCC 31101, 31102, 31103 B. mycoides ATCC 10206, CCRC 11325, 12022; B. pseudomycoides DSM 12442

Ia

1 1 1 1 1 1

1 1 1 2 3 3

1 1 1 1 2 2

1 1 2 1 1 1

A A B C D D

Ia Ia Ia Ia Ib Ib

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1 1 1 1 1 1 1 1 1 1

3 3 3 3 3 3 3 3 3 3

2 3 3 3 3 3 3 2 2 2

1 1 1 2 2 2 2 2 3 3

D E E F F F F G H H

Ib Ib Ib Ib Ib Ib Ib Ib II II

The sequences from strains in groups I, II, VI, VII, IX, XI, XII, XIII, and XVI were identical. ATCC, American Type Culture Collection; NCTC, National Collection of Type Cultures, Public Health Laboratory Service, London, England; CDC, Centers for Disease Control and Prevention; IPM, Institute of Preventive Medicine, National Defense Medical Center, Taipei, Taiwan; CCRC, Culture Collection and Research Center, Hsinchu, Taiwan; DSM, Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany. c groEL RFLP digestion proles. MboI, 1, 223 and 310 bp; MfeI, 1, 533 bp, amplimer undigested; 2, 138 and 395 bp; 3, 108 and 425 bp; PstI, 1, 109 and 424 bp; 2, 533 bp (amplimer undigested); 3, 184 and 349 bp; TspRI, 1, 136 and 397 bp; 2, 533 bp (amplimer undigested); 3, 38, 98, and 397 bp.
b

obtained from each amplied product. Comparison of sequences showed that strains within nine groups were identical to each other (Table 1). Within group I, the sequence of B. cereus ATCC 11950 was also identical to that of 12 B. anthracis strains (Table 1). Between six B. mycoides (ATCC 10206, 31101, 31102, and 31103 and CCRC 12022 and 11325) and one B. pseudomycoides (DSM 12442) strain and the other B. cereus group strains, nucleotide sequence similarity ranged from 94.2% to 95.9%. Among all B. cereus group strains studied except those seven strains, nucleotide sequence similarity ranged from 96.4 to 100%. Cloned partial sodA gene sequences of B. cereus group strains. A sequence of at least 428 bp was obtained from 21 reference B. cereus group strains. The sequence of B. cereus ATCC 21769 was identical to that of B. anthracis strain ATCC 11966. The sequences of three strains (B. cereus ATCC 33019 and CCRC 10250 and B. thuringiensis ATCC 10792) were also identical. Between B. mycoides ATCC 10206 and the other B. cereus group strains, nucleotide sequence similarity ranged

from 90.4% to 92.6%. Among all other B. cereus group strains, nucleotide sequence similarity ranged from 95.6 to 100%. Phylogenetic analysis. Based on groEL DNA sequences, the phylogenetic tree from the 51 B. cereus group strains, including reference strains and Taiwan B. cereus soil isolates, was constructed as shown in Fig. 1a. Neighbor-joining phylogenies of sodA were also constructed for the 21 reference B. cereus group strains assayed in the groEL phylogenetic analysis (Fig. 1b). The topology of the phylogenetic tree obtained from the sodA gene was in general agreement with that inferred from an analysis of the groEL gene sequences. Six B. mycoides strains and one B. pseudomycoides strain formed a very clearly dened cluster (cluster II) in the groEL tree, with good bootstrap support (100%) (Fig. 1a, Table 1). The tree constructed from the sodA gene conrmed this result (Fig. 1b). For both genes (groEL and sodA), B. anthracis and some B. cereus and B. thuringiensis strains formed a strongly supported monophyletic group (cluster Ia) (79.2 and 98.1% of bootstraps, respectively). Twelve B. anthracis, 13 B. cereus, and two B. thuringiensis

VOL. 69, 2003 TABLE 2. Specic groEL assay for 56 reference B. cereus group strains, 38 B. cereus group soil isolates, and 13 other Bacillus sp. strains
Species No. of strains PCR result with primer set ba1F ba1R

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B. anthracis B. cereus Reference strains Taiwan soil isolates B. thuringiensis B. mycoides B. pseudomycoides Other Bacillus spp. B. alcalophilus ATCC 27647 B. badius ATCC 14574 B. brevis ATCC 8246 B. circulans ATCC 4513 B. coagulans ATCC 7050 B. lentus ATCC 1840 B. lichenformis ATCC 10716 B. megaterium ATCC 14851 B. pantothenticus ATCC 14576 B. pasteurii ATCC 11859 B. polymyxa ATCC 842 B. sphaericus ATCC 14577 B. subtilis ATCC 6051

12 18 38 17 8 1 13 1 1 1 1 1 1 1 1 1 1 1 1 1

RFLP types. A single signature TspRI RFLP type was produced for the cluster II phylogenic group. MfeI and PstI RFLP analyses were capable of discriminating between the Ia and the Ib phylogenetic groups. Twelve B. anthracis strains, eight B. cereus reference strains, and four Taiwan B. cereus soil isolates were identied as both RFLP type A and groEL phylogenetic group Ia. groEL RFLP analysis revealed ve groEL RFLP types among the 38 Taiwan B. cereus soil isolates (Table 4). Four types (A, D, E, and F) identied in reference B. cereus group strains were also present in these isolates (Tables 1 and 4). Three types (B, G, and H) present in reference B. cereus group strains were not found in the Taiwan soil isolates. One type (C) was seen only in the Taiwan soil isolates. vrrA RFLP analysis of Taiwan B. cereus strains. vrrA gene PCR amplicons of approximately 1,200 bp were generated from the 38 B. cereus strains isolated in Taiwan. The restriction enzyme analysis of the PCR product cut with HinfI and MnlI produced 16 vrrA types for these isolates (Table 4). There was no similarity between the vrrA RFLP types of soil isolates and those of B. anthracis strains. DISCUSSION The 16S rRNA gene sequences determined from amplicons have contributed greatly to phylogenetic studies on eubacteria (50, 51). Like the 16S rRNA gene, groEL and sodA have been shown to be valuable tools for phylogenetic studies (20, 40, 49). In some instances, phylogenetic analyses based on groEL sequences have claried relationships that were controversial or not evident after 16S rRNA sequence comparisons (49). The topology of the groEL- and sodA-based trees was comparable in the phylogenetic trees from B. cereus group strains in our study. Three major subclusters (Ia, Ib, and II) could be identied, with an early separation between clusters I and II. In groEL trees, B. cereus group strains were grouped into two major clusters; one included six B. mycoides and one B. pseudomycoides strains, and the second included all B. cereus, B. thuringiensis, and B. anthracis and two B. mycoides strains. An identical pattern was also found for the sodA gene, and there was full correspondence among the grouping of strains according to the two genes analyzed. B. cereus, B. thuringiensis, B. mycoides, and B. anthracis have a signicant degree of genetic similarity, as demonstrated by DNA-DNA hybridization studies (26) and comparison of bac-

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strains were included in cluster Ia (Table 1). The rest of the B. cereus and B. thuringiensis strains were within cluster Ib on the basis of groEL phylogenetic analysis. The two Ib clusters in the groEL and sodA phylogenetic trees were supported by 52.8 and 79.3% bootstrap values, respectively. Specic PCR amplication of groEL gene among B. cereus group strains. Primers ba1F and ba1R amplied a single 533-bp amplicon in all 94 B. cereus group strains (Table 2). This specic PCR failed to obtain PCR products from 13 other Bacillus species (Table 2). groEL RFLP analysis of B. cereus group strains. RFLP analysis with MboI, MfeI, PstI, and TspRI was performed. Eight distinct RFLP patterns were found among the 78 B. cereus group strains examined (Table 1). Polymorphism detected by RFLP analysis of the groEL gene was consistent with the phylogenetic analysis for the B. cereus group bacteria but was more discriminative. The Ia and Ib phylogenic groups could be further differentiated into three and four groEL RFLP types, respectively. All B. cereus group bacteria had identical MboI

TABLE 3. Oligonucleotide sequences of primers used in this study

Use and primer Sequencea Reference

Universal amplication for groEL H279 H280 Universal amplication for sodA d1 d2 B. cereus group specic for groEL ba1F ba1R Amplication for vrrA GPR 6 GPR 7
a

5-GAATTCGAIIIIGCIGGIGA(TC)GGIACIACIAC-3 5-CGCGGGATCC(TC)(TG)I(TC)(TG)ITCICC(AG)AAICCIGGIGC(TC)TT-3 5-CCITA(AG)ICITA(AG)GA(AG)GCI(AG)TIGA(CT)CC-3 5-A(CT)(CT)TA(CT)TAIGC(CT)TG(AG)TCCCAIAC(CT)TC-3 5-TGCAACTGTATTAGCACAAGCT-3 5-TACCACGAAGTTTGTTCACTACT-3 5-CGTAGTTCACGAACTGCATCTATT-3 5-GATGTATCTAATGCGGCGTTAC-3

16 16 40 40 This study This study 1 1

I, nucleotide, analog inosine.

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FIG. 1. Phylogenetic trees of B. cereus group bacteria based on nucleotide sequences of groEL (a) and sodA (b). The percentage of 1,000 bootstrap replicates is indicated above the nodes. The scale bar represents 10% differences in nucleotide sequence. (a) DNA neighbor-joining phylogram inferred from the nucleotide sequences of groEL gene fragments originating from 78 B. cereus group strains, including 56 reference strains and 22 Taiwan B. cereus soil isolates. The 533-bp sequences were used. Nine groups of B. cereus group bacteria had identical sequences (Table 1). One to three representative strains in each group are represented in the gure. The tree was rooted with B. stearothermophilus as the outgroup. (b) DNA neighbor-joining phylogram inferred from the nucleotide sequences of sodA gene fragments originating from 21 reference B. cereus group strains. The 428-bp sequences were used. The tree was rooted with B. stearothermophilus as the outgroup. B. t, B. thuringiensis; B. m, B. mycoides; B. c, B. cereus; B. a, B. anthracis.

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FIG. 1Continued.

terial rRNA or ribosomal DNA at the 16S, 23S (3, 4), and 16S-23S spacer regions (6, 22). Based on their high similarity, it has been proposed repeatedly that all species of the B. cereus group be merged into a single species (3, 4, 26). However, hblA sequence analysis of 18 strains of the B. cereus group revealed two clusters of strains (43). One contained B. thuringiensis and a majority of the B. cereus strains, and the other cluster contained the B. mycoides, B. pseudomycoides, and B. weihenstephanensis strains as well as one B. cereus strain. Using amplied 16S-23S internal transcribed spacers, Daffonchio et al.

also found two main groups among B. cereus group strains (11). A phylogram generated from the groEL and sodA gene sequences of the B. cereus group correlates with the phylogenetic relationships inferred from the 16S rRNA gene sequence and supports the idea that B. cereus, B. thuringiensis, and B. anthracis should be organized into a single species (24). Evaluation of the groEL and sodA gene sequences further supports other evidence indicating two main groups among B. cereus group strains. On the other hand, Nakamura et al. (32) reported that the B.

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TABLE 4. Taiwan Bacillus cereus soil isolates (n 38) assayed for vrrA and groEL RFLP types
Strain(s) vrrA RFLP type groEL RFLP type

IPM IPM IPM IPM IPM IPM IPM IPM IPM IPM IPM IPM IPM IPM IPM IPM IPM IPM IPM

BCS BCS BCS BCS BCS BCS BCS BCS BCS BCS BCS BCS BCS BCS BCS BCS BCS BCS BCS

42, 11 15 28, 30 37 22, 33 5 27, 1, 21, 20 9 24, 6, 8, 17 29 14, 25 10 26 16 12, 13, 23 34 31 3, 4, 36, 38, 39, 40, 41 18, 19

9 11 13 14 15 10 1 4 12 16 1 2 3 1 4 5 6 7 8

A A A A A C D D D D E E E F F F F F F

mycoides group consists of two genetically distinct groups based on DNA relatedness studies. In their study, B. mycoides strain ATCC 6462 (type strain) was included in the rst group, and B. pseudomycoides was included in the second group. The second group appeared as a distinct taxon that was more distantly related to B. cereus than to the rst group (33). These results were veried in our groEL phylogenetic analysis. groEL gene sequencing separated B. mycoides strains into two groups. Cluster II grouped the six B. mycoides strains and one B. pseudomycoides. Two B. mycoides strains (including the type strain, ATCC 6462) were clustered together within cluster Ib. The sequences of these two strains were more similar to those of B. cereus and B. thuringiensis strains than to the cluster II B. mycoides strains. Eight B. cereus reference strains, two B. thuringiensis reference strains, and ve Taiwan B. cereus soil isolates were included in the Ia subcluster from the groEL phylogenetic tree. At the same time, 10 B. cereus reference strains, 15 B. thuringiensis reference strains, and 17 Taiwan B. cereus soil isolates were included in subcluster Ib. Analysis of the sodA gene further conrmed that various B. cereus and B. thuringiensis strains could be separated into two different clusters. Furthermore, in groEL RFLP studies, reference B. cereus and B. thuringiensis strains were distributed among different types. Restriction analysis of the amplied groEL fragments from B. cereus soil isolates in Taiwan also conrmed their variability. Five groEL RFLP types were found among the 38 Taiwan B. cereus soil isolates. The high genetic heterogeneity among Taiwan soil isolates was demonstrated by vrrA RFLP analysis. Phylogenetic analysis with the amplied fragment length polymorphism analysis fragment separated the Norwegian B. cereus and B. thuringiensis isolates into at least ve distinct groups (47). Helgason et al. (23) analyzed B. cereus and B. thuringiensis strains isolated from soil and found that they demonstrated very high diversity in multilocus enzyme electrophoresis genotypes. The results presented here further demonstrate that B. cereus and B. thuringiensis are highly polymorphic species. Chen and Tsen (9) reported that, except for the

cry gene or crystal protein assay, it is difcult to develop a reliable molecular method for the differentiation of B. cereus and B. thuringiensis strains. Helgason et al. reported that B. cereus and B. thuringiensis are one species, based on genetic evidence (24). Our data from groEL and sodA phylogenetic analysis also revealed that B. cereus and B. thuringiensis cannot be distinguished easily, apart from the entomocidal genotype and phenotype. In contrast to the lack of species grouping by B. cereus and B. thuringiensis isolates, the 12 strains of B. anthracis had identical groEL sequences, which conrmed that it is a uniform and clonal species. This is consistent with the previously described monomorphic nature of this species (27, 28, 29). We also found several reference strains (including emesis-inducing strain NCTC 11143, diarrhea-inducing strain ATCC 49064, and B. cereus ATCC 11950), several Taiwan soil isolates, and 12 B. anthracis strains were included in the same subcluster from the groEL phylogenetic tree. The similarity value was high (99.3%) between the emetic strain (NCTC 11143) and B. anthracis in the groEL sequences analysis. Ash et al. (3) reported that the sequence of B. cereus emetic strain NCTC 11143 and B. anthracis were identical for a continuous 1,446 bases of 16S rRNA. In addition, a high sequence similarity value (99.9%) was obtained for the 2,889-nucleotide regions of the 23S rRNA gene of this strain and B. anthracis (4). B. cereus strain ATCC 11950 also had a partial groEL sequence that was identical to that of the 12 B. anthracis strains. Patra et al. (36) proposed that a 277-bp fragment (BA813) is useful for B. anthracis identication, and this fragment was absent from all the other Bacillus species examined. The BA813 marker was present in B. cereus strain ATCC 11950 (data not shown), which indicated the high similarity between this strain and B. anthracis. The results presented in this study demonstrated that B. anthracis is genetically closely related to several B. cereus food poisoning and environmental strains. Perhaps the difculty in differentiating B. anthracis strains is because B. anthracis is actually a subspecies of the much more polymorphic B. cereus lineage (7). The phenotypic differences may be altered by horizontal transfer of plasmids (24). Enterotoxigenic B. cereus, which causes acute gastroenteritis after the consumption of contaminated foods, has been known to produce emetic and diarrheal toxins (19). Strains from the other B. cereus group species B. thuringiensis, B. mycoides, and B. weihenstephanensis are also potentially enterotoxic, as they possess the bceT gene and genes in the hemolysin and nonhemolytic enterotoxin operons for the production of enterotoxins (21, 43). Thus, all B. cereus group strains may be potentially toxigenic, and the detection of these organisms in foods is important. The use of nucleic acid amplication by PCR has applications in many elds, especially for the rapid identication of bacteria. In this study, we were able to identify all B. cereus group strains analyzed. This PCR procedure is further enhanced when it is combined with RFLP analysis, which allows identication and discrimination of the B. cereus group bacteria. All B. cereus group isolates had the same MboI RFLP type. A single signature TspRI RFLP type was produced for cluster II B. mycoides and B. pseudomycoides strains. RFLP with MfeI or PstI classied all B. cereus and B. thuringiensis into two groups. Although other virulence factors must be determined

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to conrm the pathogenicity of B. cereus group bacteria, this simple PCR method has the potential not only to rapidly identify B. cereus group in culture, but also to detect and differentiate B. cereus group strains in food samples. The PCR RFLP test could not differentiate B. anthracis from other B. cereus group bacteria. However, sequencing of the 533-bp groEL gene could identify B. anthracis. Only one strain (B. cereus ATCC 11950) out of 56 reference B. cereus group strains and 38 Taiwan Bacillus cereus soil isolates had the same sequence as the B. anthracis strains. Furthermore, on the basis of the sequenced groEL genes, we designed three oligonucleotide probes for identication of the B. anthracis strains. The initial results showed that B. anthracis strains can be identied when performing PCR-enzyme-linked immunosorbent assay analysis with these probes (data not shown). In conclusion, the use of phylogenetic markers besides 16S rRNA is a useful supplement to differentiate B. cereus group bacteria. In this work, we described a method for classifying B. cereus group bacteria with partial groEL gene sequences as a phylogenetic marker. With our approach, we found that B. anthracis strains are highly related to several B. cereus strains. The results conrmed previously reported ndings that B. anthracis is very homogeneous and that its apparent relatedness to B. cereus and B. thuringiensis is highly dependent on the strains studied (20). For the detection of B. cereus group cells, we designed PCR primers from groEL sequences. In addition, RFLP analysis of PCR-amplied groEL allowed separation of B. cereus group strains into different types.
ACKNOWLEDGMENT We thank Chung-Chih Liang for excellent technical assistance in the laboratory.
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