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Applied and Environmental Microbiology, February 2006, p. 1569-1578, Vol. 72, No. 2
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.2.1569-1578.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Multiple-Locus Sequence Typing Analysis of Bacillus cereus and Bacillus thuringiensis Reveals Separate Clustering and a Distinct Population Structure of Psychrotrophic Strains

Alexei Sorokin,1* Benjamin Candelon,1,{dagger} Kévin Guilloux,1 Nathalie Galleron,1 Natalia Wackerow-Kouzova,1,{ddagger} S. Dusko Ehrlich,1 Denis Bourguet,2,§ and Vincent Sanchis2

Génétique Microbienne, INRA, Domaine de Vilvert, 78352 Jouy en Josas cedex,1 Génétique Microbienne et Environnement, INRA La Minière, 78285 Guyancourt cedex, France2

Received 6 September 2005/ Accepted 8 November 2005


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ABSTRACT
 
We used multilocus sequence typing (MLST) to characterize phylogenetic relationships for a collection of Bacillus cereus group strains isolated from forest soil in the Paris area during a mild winter. This collection contains multiple strains isolated from the same soil sample and strains isolated from samples from different sites. We characterized 115 strains of this collection and 19 other strains based on the sequences of the clpC, dinB, gdpD, panC, purF, and yhfL loci. The number of alleles ranged from 36 to 53, and a total of 93 allelic profiles or sequence types were distinguished. We identified three major strain clusters—C, T, and W—based on the comparison of individual gene sequences or concatenated sequences. Some less representative clusters and subclusters were also distinguished. Analysis of the MLST data using the concept of clonal complexes led to the identification of two, five, and three such groups in clusters C, T, and W, respectively. Some of the forest isolates were closely related to independently isolated psychrotrophic strains. Systematic testing of the strains of this collection showed that almost all the strains that were able to grow at a low temperature (6°C) belonged to cluster W. Most of these strains, including three independently isolated strains, belong to two clonal complexes and are therefore very closely related genetically. These clonal complexes represent strains corresponding to the previously identified species Bacillus weihenstephanensis. Most of the other strains of our collection, including some from the W cluster, are not psychrotrophic. B. weihenstephanensis (cluster W) strains appear to comprise an effectively sexual population, whereas Bacillus thuringiensis (cluster T) and B. cereus (cluster C) have clonal population structures.


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INTRODUCTION
 
The Bacillus cereus group consists of gram-positive spore-forming bacteria with an impact on human activities due to their pathogenic properties. Strains capable of producing large amounts of entomopathogenic toxins are designated Bacillus thuringiensis and are commonly used as effective biopesticides. Strains producing emetic toxins or enterotoxins and causing food poisoning following the ingestion of contaminated food are usually classified as Bacillus cereus. Strains of the closely related species Bacillus anthracis cause a lethal disease (anthrax). The recently identified species Bacillus weihenstephanensis (20) is characterized by an ability to grow at low temperatures (below 8°C). Bacillus mycoides forms colonies with a specific form due to rhizoidal growth. Several strain classification studies have shown that it is difficult to separate B. cereus and B. thuringiensis into two clusters (2, 3, 6, 13-16, 25, 32). This suggests that it is not possible to distinguish between these two species on the basis of neutral genome sequences that are not related to the production of entomopathogenic toxins. This situation may arise from intensive exchange of genetic material between the two species in their natural habitat. Formally, B. thuringiensis may be defined as phenotypically similar to B. cereus but with a naturally acquired ability to synthesize large amounts of entomopathogenic toxin, forming parasporal crystals visible under the microscope in sporulating cells. A collection of B. thuringiensis and B. cereus strains was recently isolated from the soil of a forest near Versailles (close to Paris, France) (33). The strains of this collection were classified as B. cereus or B. thuringiensis based on their ability to produce parasporal toxin. Multiple-locus enzyme electrophoresis (MLEE) showed that the populations of B. cereus and B. thuringiensis living in the same soil were genetically distinct and diverged to a greater extent than strains of the same species isolated from geographically different locations. This suggests that ecological separation, which is probably the main force behind separation and cohesion for bacterial genetic clustering, is stronger than the potential of these bacteria to exchange genetic material. In this case, genetic exchange is mediated by plasmids encoding the crystal proteins, enabling the bacterium to occupy the ecological niche of an insect pathogen.

We addressed similar questions concerning the fine genetic structure of this collection of strains using a different experimental approach, because the MLEE method is insufficiently precise for studies of the fine genetic structure of bacterial populations. Furthermore, MLEE generates data that cannot easily be compared with data from independent studies and data for other strain collections. We therefore chose to use the multiple-locus sequence typing (MLST) approach (22). This approach involves sequencing short (300- to 700-bp) regions of several selected genes randomly distributed over the bacterial chromosome. The genes are usually chosen such that structural changes reflect vertical bacterial evolution, with no major influence of specific niche-related selection (housekeeping genes). We investigated the following questions: (i) whether toxin-producing and non-toxin-producing populations in the same soil are different enough genetically for formal clustering into two separate clusters; (ii) whether the strains of the Bacillus cereus group collected from small samples of soil presented genetic evidence for active sexual exchange within local populations; (iii) what was the natural potential of different populations to display genetic homogeneity. We tackled these questions by studying natural populations of these bacteria isolated from different samples and under different conditions.


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MATERIALS AND METHODS
 
Strains and growth conditions.
All the strains used in this study are shown in Fig. 1. The strains for which experimental sequence data were obtained in this study were given designations beginning with the letters KB. The 115 strains labeled KBAnn, KBBnn, and KBCnn are from the Versailles Collection, which has been described elsewhere (33). They were isolated from the soil of a forest at La Minière near Versailles, France (Ile-de-France region) on 6 December 2000. B. thuringiensis strains were distinguished from B. cereus strains by light microscopic detection of parasporal crystals (33). We also applied MLST analysis to five psychrotrophic B. weihenstephanensis strains—WSBC10204, WSBC10206, WSBC10297, WSBC10311, and WSBC10315 (20, 27) (Fig. 1, KB008 to KB012, respectively)—obtained from S. Scherer (Germany); five strains of B. cereus—41, F4810/72, F0837/76, F2038/78, and ATCC 6464—reported to have smaller than normal chromosomes (4) (KBBI1 to KBBI5, respectively), obtained from A.-B. Kolsto (Norway); a pathogenic B. cereus isolate, NVH 391-98 (21) (KB007), from vegetable puree, obtained from D. Lereclus (France); and B. cereus strain BGSC 6A1(1) (KB002) and four strains of B. thuringiensis, BGSC 4H2, BGSC 4D1, ATCC 35646, and BGSC 4Q7 (5, 8, 9, 30) (KB003 to KB006, respectively), from public collections. We also included the sequences of the loci considered from the entirely sequenced strains B. cereus ATCC 14579 (17) (KB001), B. cereus ATCC 10987(28), and B. anthracis Ames (29) in our analysis.


Figure 1
Figure 1
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  		FIG. 1. UPGMA-based dendrogram and BURST-based clonal complexes of the allelic profiles of the 134 strains used in this study. Each strain name is followed by the strain origin marker (consisting of the letters Bt, Bc, Ba, or Bw [phenotypically defined B. thuringiensis, B. cereus, B. anthracis, or B. weihenstephanensis, respectively], or BtF or BtS [strains from the Versailles Collection originating from deep in the forest or the forest edge], and a number indicating the soil sample [33]), the sequence-based strain cluster designation, the cold-growth phenotype (M, P, I, or N [see Materials and Methods for an explanation]), the ST, and the corresponding allelic profile. The allelic profile, in parentheses, contains arbitrary allelic numbers for the clpC, dinB, gdpD, panC, purF, and yhfL loci, respectively. Most of the strains fall into clonal complexes (or groups, designated G1, G2, etc.) of closely related STs, defined as groups of STs in which every ST shares at least four of six identical alleles with at least one other ST in the group. Singletons are STs not belonging to the clonal complexes. Groups marked with asterisks have ancestral allelic profiles identified by START. Relationships between single-locus variants (red), double-locus variants (blue), and the ancestor are indicated by concentric circles or connecting lines of the corresponding color, generated by START (18). (A, B, and C) Allelic profile trees for the T, W, and C clusters, respectively. Green vertical bars indicate clonal complexes of psychrotrophic strains.

All strains were cultured overnight on Luria-Bertani agar plates at 30°C, as previously described (16), and stored at room temperature until MLST analysis. The cells (approximately 107) were picked by toothpick into 300 µl of water and resuspended by vortexing, and 3 µl of the resulting suspension was used for PCR. The ability of strains to grow in the cold was tested by streaking bacteria from plates incubated overnight at 30°C onto fresh agar plates. These plates were then incubated for 14 days at 6°C. Growth was assessed by eye, with the density of bacterial growth compared to that at 30°C and expressed as a percentage of this maximal growth. This procedure was repeated three times, and we estimated the statistical error of this method of growth estimation at 30%. The cold-growth status of strains was designated as follows: P (for psychrotrophic) if growth exceeded 70% of the maximum, M (mesophilic) if less than 30% of the maximum level of growth was observed, I for intermediate growth, and N for strains not tested (see Fig. 1).

PCR amplification and sequencing.
We carried out PCR with the Expand Long Template PCR system (Roche Diagnostics) to obtain templates for sequencing. The cycling program was as follows: 94°C for 5 min; 12 cycles of 94°C for 10 s, 55°C for 10 s, and 65°C for 12 min; 24 cycles of 94°C for 10 s, 55°C for 10 s, and 65°C for 12 min in the first cycle, increasing by 15 s with each subsequent cycle. The mixture was subjected to a final extension step for 10 min at 72°C. PCR products were treated for 1 h at 37°C with exonuclease I and shrimp alkaline phosphatase (USB Corporation). Sequencing was performed with an ABI PRISM sequencing kit (Applied Biosystems). Sequencing products were precipitated in ethanol and analyzed with an ABI 3700 sequencer (Applied Biosystems).

MLST.
We used multiple-locus sequence typing to compare strains. Alleles at six unlinked loci were identified by sequencing of 350- to 700-bp internal fragments of the genes, and the sequence type (ST) was defined by the string of allele numbers at the six loci. The six genes selected for characterization of the diversity of strains by MLST (22) and the corresponding primers are listed in Table 1. They are evenly distributed over the chromosome of the entirely sequenced strain B. cereus ATCC 14579 (17), referred to here as KB001. The alternative primers used for amplification and sequencing of the strains of the W cluster were based on the sequences of flanking regions of the selected loci for strains KB008, KB009, and KBAB4, considered representative of this cluster. These sequences are available from NCBI under accession numbers DQ301422 to DQ301433.


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  		TABLE 1. Primers used for PCR amplification

The sequences from each strain were assembled and consensus sequences generated with xbap software (7). The consensus sequences were compared using the multiple alignment program CLUSTALX 1.8 (31), and phylogenetic trees were generated with NJplot software (24). START software was used for the analysis of allelic data in the MLST context (18). The program decoupe_msf, used for the elimination of identical bases from closely related aligned sequences, was kindly provided by J.-M. Batto (Génétique Microbienne, INRA). The sequences were deposited in the NCBI database and can also be used for BLAST analysis at spock.jouy.inra.fr (click on "Links" and "BaCerId" in the site menu).

Linkage analysis and prediction of clonal complexes.
We assessed the correlation between alleles in the population and used this information to determine clonal status (23) by calculating the index of association (IA) with START software (18). Clonal complexes or groups of strains, defined as strains with no more than two different alleles, and the ancestral strains were predicted using the BURST algorithm implemented in START.

Nucleotide sequence accession numbers.
The sequences determined for the 115 strains studied were deposited in the NCBI database under accession numbers DQ296198 to DQ296463 and DQ300927 to DQ301422.


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RESULTS
 
The phylogeny based on sequence alignment reveals three major strain clusters.
Six genes—clpC, dinB, gdpD, panC, purF, and yhfL—were systematically sequenced for 115 strains of the Versailles Collection, including 61 and 54 strains that had previously been identified as B. cereus and B. thuringiensis, respectively (33). These genes were also sequenced for 19 reference strains, as described in Materials and Methods. Phylogenetic analysis based on the sequences of individual loci (not shown) or concatenated sequences and the neighbor-joining method implemented in CLUSTALX (25) indicated that most strains could be assigned to one of three clusters: C, T, and W (Fig. 2). The set of oligonucleotides selected on the basis of available sequences for KB001 and B. anthracis Ames (17, 29) gave no PCR amplification for many cluster W strains (not shown). We therefore amplified entire regions containing the selected genes for strains KB008, KB009, and KBAB4, which are representative of this cluster, using primers corresponding to the coding regions of the nearest conserved genes. We obtained PCR fragments of 1.5 to 5 kb. Sequencing of these fragments by primer walking provided the necessary information for the selection of an alternative set of oligonucleotides, making it possible to amplify the loci of cluster W strains in a reproducible manner. Table 2 shows the statistical characteristics of the entire sequencing data set and a comparison of the characteristics of the three clusters. The assignment of individual strains to these clusters is shown in Fig. 1. Four strains from the forest soil collection could not be assigned to any of the three clusters; these strains were assigned the cluster name "X." Strain KB007 was unusual in that it had highly divergent alleles for three genes (clpC, gdpD, and yhfL) but could clearly be assigned to cluster W based on analysis of the other three genes (dinB, panC, and purF).


Figure 2
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  		FIG. 2. Concatenated sequence-based phylogenetic tree. Three clusters containing most of the strains are designated by the letters C, T, and W. Each strain name is followed by the ST number and the number of identical strains corresponding to this ST. Approximate locations of clonal clusters (or groups) are indicated, including G1, G2, and G8, corresponding to the lineages Tolworthi, Sotto, and Kurstaki identified by Priest et al. (26). The small cluster X, containing strains KBAE4, KBBC4, KBCF5, and KBCC8, is not labeled. Bc10987, B. cereus 10987; BAames, B. anthracis Ames.


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  		TABLE 2. Genetic diversity in the loci studied and in different strain clusters

The six genes sequenced therefore gave consistent separation of the strains into three major, nonoverlapping clusters. We carried out allele profile analysis for each of these clusters, both separately and together. This separation into clusters was also used as the basis for a separate calculation of indices of association.

STs and clonal complexes.
Each unique sequence of a gene was regarded as an allele and was assigned an arbitrary allele number. The set of allele numbers for a given strain represents an allelic profile or sequence type, also arbitrarily numbered. Figure 1 shows dendrograms constructed using the unweighted-pair group method using average linkages (UPGMA), with allelic profiles or STs. The strains were resolved into 93 STs, 80 of which were identified only once. The most common, ST-93, was identified 12 times, whereas two others, ST-54 and ST-57, were detected in 11 and 8 isolates, respectively, and 10 STs corresponded to 2 or 3 strains. Most of the 115 isolates of the Versailles Collection and the 19 additional strains analyzed in this work were grouped into 10 clonal complexes, with the isolates of a given complex identical at four, five, or six loci (Fig. 1). The clonal complexes for which the ancestral ST was successfully identified with START software are represented in the form of concentric circles. The ancestral ST is shown in the central circle. STs differing by only one (single-locus variants) or two (double-locus variants) loci are shown separated from the ancestor by one or two circles, respectively. This formalism of the presentation of very close phylogenetic relationships between bacterial strains, and the concept of clonal complexes, which is very useful for the interpretation of MLST data, were suggested by Feil et al. (11). START also uses color coding to show more complex relationships between STs (see the legend to Fig. 1).

The three clusters—C, T, and W—consisted of 15, 55, and 41 strains, respectively, isolated from forest soil. The significance of this formal cluster assignment is made clear by the inclusion in our data set of sequences from independently isolated strains with established phenotypic characteristics. Several B. thuringiensis strains—Bacillus thuringiensis serovar israelensis ATCC 35646 (KB005), B. thuringiensis serovar israelensis BGSC 4Q7 (KB006), Bacillus thuringiensis serovar kurstaki BGSC 4D1 (KB004), and Bacillus thuringiensis serovar canadensis BGSC 4H2 (KB003)—and two strains of B. cereus—ATCC 14579 (KB001) and BGSC 6A1 (KB002)—appeared to belong to cluster T. Moreover, almost all the strains of our forest collection found to synthesize parasporal crystal toxin protein (33) were also found in this cluster (Fig. 1A). These strains are therefore genetically different from the strains in clusters C and W even in the absence of genes encoding entomopathogenic toxins, as in the case of strains KB001 and KB002. In our forest strain collection, only one group (or clonal complex), designated G8, contained a mixture of strains assigned to B. cereus and B. thuringiensis based on phenotype. Cluster X (Fig. 1C) was also a mixed group. It contained both phenotypic classes and was not identified as a clonal complex, because allelic profiles differed at more than two loci. In total, we detected five clonal complexes (G1, G2, G6, G7, and G8) for cluster T, four of which had an identifiable ancestral strain (Fig. 1A). These clonal complexes displayed strain relationships different from those for cluster W (Fig. 1B). Most of the cluster T strains had identical STs or differed from the ancestral strain by only one allele. One possible reason for this is that strains may be clones, and such findings may result from an artifact of strain isolation. All precautions were taken to avoid this (33), and at least two other lines of evidence suggest that this is not the case: (i) only five strains with the same phenotype were isolated from the same soil sample, and (ii) if there were clones, we would expect similar population structures for the three clusters (T, C, and W), whereas these clusters were actually found to have different population structures.

The strains that did not synthesize parasporal crystals detectable by light microscopy belonged to two major clusters: W and C. Most of the cluster W strains could be combined into two clonal complexes (G3 and G5), demonstrating their close genetic relationship (Fig. 1B). Only one genetically different group, designated G10, was identified in cluster W, although six strains, including the pathogenic strain KB007 and the independently isolated psychrotrophic strain KB010 (27), would clearly form another clonal complex if more strains were analyzed (Fig. 1B).

Cluster C contained about one-third of all the tested strains of the Versailles Collection identified as B. cereus on the basis of phenotype (Fig. 1C). This cluster also contained pathogenic B. anthracis Ames, B. cereus ATCC 10987, and emetic strain KBBI2. Two psychrotrophic strains, KB011 and KB012, isolated independently (27), also belonged to this cluster, although testing of these and all other strains of this cluster (see below) showed a tendency to grow less well in the cold. Indeed, our solid-medium test at 6°C characterized strains KB010, KB011, and KB012 as mesophilic, whereas they were classified as psychrotrophic based on culture in liquid medium at 8°C (see below). Cluster C contained fewer strains than cluster W, accounting for the lack of isolation of rare strains closely related to KB011 and KB012. We identified two clonal complexes in this cluster, G4 and G9, but were unable to identify the ancestral STs.

Relationship between strain clusters and psychrotrophy.
The ability to grow in the cold is not an intrinsic property of all B. cereus or B. thuringiensis strains. When the Versailles Collection was established, psychrotrophy was not suspected in many strains and was not systematically tested (33). However, the close genetic relationship between strains from clonal complex G3 of cluster W and the psychrotrophic type strains KB008 and KB009 (20) suggests that many strains of this clonal complex may be able to grow at low temperatures. We therefore systematically tested all strains that did not synthesize detectable parasporal crystals (clusters W and C) and most of the strains that did produce such crysals (cluster T) for the ability to grow at 6°C on solid medium. These results are shown on Fig. 1. In this strict test (ability to grow at 6°C), most of the cluster W strains were found to be psychrotrophic. Almost all these strains were assigned to two clonal complexes—G3 and G5—containing 22 and 7 strains, respectively. These findings indicate that the strains that are able to grow in the cold are closely related genetically. Group G10 of cluster W, which was genetically different, contained two mesophilic strains. Genetic characterization of five independently isolated psychrotrophic strains (20, 27), designated KB008 to KB012, revealed that KB008 belonged to the main psychrotrophic clonal complex of the Versailles Collection and that KB009 was closely related. However, three other independently isolated psychrotrophic strains, KB010, KB011, and KB012, did not seem to be closely related to the strains in this collection. KB011 shares two identical alleles with several strains of cluster C (group G9), but several forest strains from this cluster (e.g., KBAC5, KBCA4, and KBCB1) grew poorly in the cold in our test, and others (e.g., KBCA5, KBCE4, and KBCG5) did not grow in the cold at all. In this strict test of growth in the cold (6°C), strains KB010, KB011, and KB012 were actually classified as mesophilic (Fig. 1). However, these strains grew to a high density (optical density at 600 nm, 3) in liquid medium at 8°C (not shown). The 14 strains of the W cluster, phylogenetically identified as singletons not belonging to any clonal cluster, differed in their ability to grow in the cold. Nine of these strains grew reproducibly at 6°C, three did not grow at this temperature, and growth was not reproducible for the other two (Fig. 1). We therefore conclude that, in the collection tested, only the genetically closely related G3 and G5 clonal complexes contain multiple strains with a high potential for growth in the cold.

Statistical recombination tests and the population structure of the B. weihenstephanensis and B. thuringiensis forest soil community.
The large number of strains from the T and W clusters made it possible to compare the population structures of these clusters. Visual comparison of the topologies of the phylogenetic trees constructed for the T and W clusters (Fig. 1A and B) indicated differences in the population structures of the strains in these two clusters, with presumably clonal population structures for cluster T and effectively sexual (or panmictic) population structures for cluster W. Several clonal complexes of cluster T (G1, G2, and G7) contained a number of strains with the same STs (Fig. 1A). In contrast, the topologies of trees for clonal complexes G3, G5, and G10 differed (Fig. 1B), since only a few strains had the same allelic profiles. The topology of clonal complex G9 in cluster C also seems to resemble those of the clonal complexes of the W cluster more closely than those of other clonal complexes of the C cluster.

A quantitative parameter for distinguishing between different bacterial population structures, the index of association (IA), was proposed by Maynard Smith et al. (23). Differences in this parameter, calculated for the whole population and its parts, in particular those containing STs taken as units, can be used to distinguish between completely sexual (IA = 0), epidemic, sexual at fine scale, and completely clonal (IA >> 0 for all parts of the population) populations. For the forest strains used here, the index of association was higher than zero (IA, 2.917 [n = 115 strains]) and similar to that calculated for all strains (IA, 2.815 [n = 134]). A similar value was obtained if the strains from deep in the forest or from the forest edge were considered separately (IA, 2.807 [n = 81] and 3.861 [n = 34], respectively), presumably indicating a high correlation of sequenced alleles and the absence of intensive recombination in the population as a whole. However, the index was much lower if only strains from the W cluster were considered (IA, 0.593 [n = 41]) but remained high for the T and C clusters (IA, 4.257 [n = 55] and 2.705 [n =15], respectively). For these two clusters, the index for all clonal complexes, identified by START, exceeded 2.5. In contrast, groups G3 and G5 had lower indices (IA, 0.151 [n =20] and 0.243 [n = 7], respectively), indicating effective sexual exchange of alleles, decreasing the correlation between them. Inclusion of the 19 independently isolated strains in the analysis had no significant effect on these findings.

Recombination-to-point mutation ratio in B. weihenstephanensis and B. thuringiensis.
As reported for other bacterial populations (12), we estimated the ratio between allelic variations due to point mutations and those due to recombination events. We used the approach described by Feil et al. (11). Allelic profiles from clonal complexes in clusters W and T and sequence differences between alleles were analyzed to determine whether the alleles differed from their ancestors by a single nucleotide change, indicating that they were generated by mutation or recombination, or displayed multiple changes, indicating that they were generated by recombination alone (11). The corresponding data are presented in Table 3. For cluster W, the ratio of multiple- to single-change alleles was 13 to 7 (1.9:1), and for cluster T it was 5 to 9 (0.6:1). The corresponding estimates for the probability of changes per site were 15:1 and 3.5:1, respectively, for the W and T clusters. These figures are smaller than those reported for Neisseria meningitidis (100:1), Streptococcus pneumoniae (61:1), and Staphylococcus aureus (24:1) (12), especially for the T cluster population. The higher frequency of recombinational allele evolution in B. weihenstephanensis (W cluster) is consistent with the effectively sexual nature of this community, contrasting with the clonal nature of B. thuringiensis (cluster T).


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  		TABLE 3. Allelic variants within clonal complexes (groups) or sequence clusters


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DISCUSSION
 
We describe here the characterization of a collection of B. cereus group strains isolated from the soil of a forest near Versailles (close to Paris, France) by multiple-locus sequence typing. This study aimed to provide insight into the degree of genetic separation of B. cereus and B. thuringiensis, which have been defined on the basis of ability to synthesize parasporal toxin proteins. We were able to separate the strains studied into three major clusters: C, T, and W. The existence of such a distinct third cluster (W) of strains in this collection was unexpected. Psychrotrophy tests showed that this cluster corresponds to the recently defined species B. weihenstephanensis (20). However, not all of the strains in this cluster were psychrotrophic, and some strains previously assigned to this species were found to belong to cluster C. These three clusters appeared to have different population structures, as defined based on natural effective exchange of genetic information. Genetic exchange in the bacterial population was determined by calculating indices of association for different groups of strains and by estimating the ratio of recombination to point mutations for the two strain clusters. Cluster W was found to be effectively sexual, with frequent exchanges between strains, whereas clusters C and T appeared to be clonal.

The close relationship between forest collection strains from cluster W and the independently isolated psychrotrophic type strains KB008 and KB009 also indicates that this cluster corresponds to B. weihenstephanensis (20). However, not all of the strains assigned to this cluster on the basis of sequencing data were psychrotrophic. Thus, strains cannot be unambiguously assigned to this species based solely on the results of psychrotrophy testing. Clonal complex G10, identified here, contained three strains, only one of which was psychrotrophic. Conversely, strains KB011 and KB012, previously identified as B. weihenstephanensis (20), were assigned to cluster C and therefore to another species, B. cereus. It is difficult to apply the concept of "species" to the bacterial collection studied here. Since the use of this concept relates to the possibilities of lateral gene exchange, it is important to estimate the frequencies of such exchanges in the clonal complexes detected. We estimated the relevant parameters for the populations of clusters T and W and found differences between these populations. We found that clonal complexes G3 and G5 contained most of the psychrotrophic strains, which displayed frequent genetic exchanges. These strains may therefore act as an efficient source of genetic alleles for psychrotrophy. Future studies should test this hypothesis and investigate the molecular mechanisms involved in this gene transfer.

The work reported here corresponds to one of four independently initiated efforts to provide an MLST scheme for the B. cereus group (16, 26, 31). The scheme proposed by Helgason et al. (16) was also applied to a group of seven emetic strains, confirming their high genetic homogeneity (10). Although the four strain collections used in these studies were different, all four studies have reported the almost unambiguous assignment of strains to three major clusters. After calculating the IA for the entire collection and detecting lineages in the clusters corresponding to our C and T clusters, Helgason et al. (16) and Priest et al. (26) concluded that the whole Bacillus cereus group has a clonal or weakly clonal population structure. Ko et al. (19)discovered an unusual feature of the plcR gene, presumably related to its horizontal transfer. Our work is original in that we studied a large number of strains isolated from a very limited geographical location (a forest near Versailles). We did not expect to find such a large number of B. weihenstephanensis strains in our collection. Our collection represents a random set of strains from each cluster (C, T, and W). The population structure detected is therefore representative of the soil, limited to the place and time of sampling.

It is currently difficult to determine which of the MLST schemes produced for the Bacillus cereus group is the most appropriate. None of these studies has identified reproducibly performing loci in the chromosome region between 1,800 and 3,800 kb, known to be the most variable between strains of this group (17, 29). Nevertheless, since several strains were common to different studies, a correspondence can be established between the various clusters, clades, lineages, or clonal complexes detected. Our cluster W clearly corresponds to clade 3 of Priest et al. (26) and to cluster I of Helgason et al. (16). Similarly, cluster C corresponds to clusters 1 and III, and cluster T corresponds to clusters 2 and II, of these previous studies. Our study represents the most extensive work on the W cluster, with 41 strains of this cluster characterized versus only 6 and 22 in the studies of Priest et al. (26) and Helgason et al. (16), respectively. This made it possible to detect clonal complexes G3, G5, and G10 (Fig. 1 and 2) in this cluster. No such detection of clonal complexes was possible in previous studies, due to the small number of strains from this cluster. We estimated indices of association, which appeared to be close to zero, and the recombination/mutation ratio, revealing the panmictic population structure of cluster W. This finding does not conflict with the conclusion that the whole Bacillus cereus group is clonal, since, if we consider the three clusters together, or T and C separately, they are clonal in terms of these parameters.

Three of the clonal complexes we detected, G1, G2, and G8, correspond to the lineages Tolworthi, Sotto, and Kurstaki, identified by Priest et al. (26) (Fig. 2). An analysis of several identical strains revealed correspondence between phylogenetic trees constructed independently for different strain collections, which can therefore partially replace the use of the same MLST scheme. Thus, the Anthracis lineage (26), extensively presented in the papers of Helgason et al. (16) and Ko et al. (19), and the Cereus III lineage (26), which includes emetic strains studied by Ehling-Schulz et al. (10), correspond to two other clonal complexes. However, in these two cases, MLST is not sensitive enough for determination of the internal structure of these clonal complexes, since the sequences for almost all strains studied are the same.

The panmictic structure of the psychrotrophic strain community reported here raises questions about the mechanism underlying this intensive exchange of DNA in the natural population of B. weihenstephanensis. This high level of genetic exchange may be related to the fact that this bacterium is less clearly pathogenic than B. cereus or B. thuringiensis. It is possible that B. weihenstephanensis is really a benign soil bacterium with high genetic exchange potential. The appearance of a new ecological niche, mediated by the presence of insect or animal bodies, may thus have led to the emergence of a new pathogenic lineage. Sequencing of the genomes of a few strains of the W cluster and comparison with the genomes of strains in the C and T clusters may make it possible to determine whether this is actually the case.


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ACKNOWLEDGMENTS
 
We thank D. Zeigler, A.-B. Kolsto, D. Lereclus, A. Lapidus, and S. Scherer for kindly providing bacterial strains. We greatly appreciate the assistance of J.-M. Batto, A. Bolotin, and B. Quinquis (Genetique Microbienne, CRJ, INRA) in sequencing and computer programming. We thank Alex Edelman & Associates for editing the manuscript.

N.W.-K. held a short-term INRA fellowship.


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FOOTNOTES
 
* Corresponding author. Mailing address: Génétique Microbienne, INRA, Domaine de Vilvert, 78352 Jouy en Josas cedex, France. Phone: (33) 1 34 65 27 24. Fax: (33) 1 34 65 25 21. E-mail: sorokine{at}jouy.inra.fr. Back

{dagger} Present address: Section of Infectious Diseases, Department of Internal Medicine, Yale University School of Medicine, 1 Gilbert Street, TAC S140, New Haven, CT 06510. Back

{ddagger} Present address: Agrophysics Research Institute, Grazhdansky pr. 14, 195220 St. Petersburg, Russia. Back

§ Present address: Centre de Biologie et de Gestion des Populations (CBGP), Campus International de Baillarguet, CS 30 016, 34988 Montferrier/Lez cedex, France. Back


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Applied and Environmental Microbiology, February 2006, p. 1569-1578, Vol. 72, No. 2
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.2.1569-1578.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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