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Journal of Clinical Microbiology, April 2007, p. 1274-1277, Vol. 45, No. 4
0095-1137/07/$08.00+0     doi:10.1128/JCM.02224-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

A New Phylogenetic Cluster of Cereulide-Producing Bacillus cereus Strains{triangledown}

Maria Vassileva,1 Keizo Torii,1 Megumi Oshimoto,1 Akira Okamoto,1 Norio Agata,2 Keiko Yamada,1 Tadao Hasegawa,3 and Michio Ohta1*

Department of Bacteriology, Nagoya University Graduate School of Medicine, Nagoya, Japan,1 Nagoya City Public Health Research Institute, Nagoya, Japan,2 Department of Microbiology, Nagoya City University, Nagoya, Japan3

Received 31 October 2006/ Returned for modification 18 December 2006/ Accepted 8 February 2007


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Phenotypic and molecular studies have established that cereulide-producing strains of Bacillus cereus are a distinct and probably recently emerged clone within the Bacillus population. We analyzed a set of B. cereus strains, both cereulide producers and nonproducers, by multilocus sequence typing. Consistent with earlier reports, nonproducers demonstrated high heterogeneity. Most cereulide-producing strains and all flagellar antigen type H1 strains were allocated to the known sequence type of exclusively emetic B. cereus strains. Several cereulide-producing strains, however, were recovered at a new phylogenetic location, all of which were serotype H3 or H12. We hypothesize that the group of cereulide producers is diversifying progressively, probably by lateral transfer of the corresponding gene complex.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Bacillus cereus is often associated with food contamination, food spoilage, and food poisoning. The clinical presentation takes one of two forms, emetic and diarrheal. It is well established that the emetic type of food poisoning is associated with a single heat-stable exotoxin, cereulide (2, 8, 22). Diarrheal food poisoning, on the other hand, can be caused by several thermolabile enterotoxins (6).

Agata et al. have shown that most cereulide-producing strains belong to serovar H1, with the exception of a few that belong to serotypes H3 and H12 (1). Thus, cereulide producers appear to be a homogeneous group. Phenetic and molecular typing studies have established that cereulide-producing B. cereus strains belong to a highly homogeneous cluster (9), which was also suggested by earlier reports of identical ribopatterns of 16S and 23S rRNA sequences from emetic strains (5, 15, 17). Multilocus sequence typing (MLST) has assigned all analyzed cereulide-producing strains, isolated from cases of both food poisoning and the environment, to single-sequence type (ST) 26 (16). Recently, however, a few cereulide-producing strains have been reported to possess unique ribopatterns and to demonstrate a variation in the housekeeping gene adk (4).

This unexpected finding leaves open the question of how widely the capacity for emetic toxin production is distributed among B. cereus strains. Should these findings be considered minor discrepancies, or is heterogeneity among cereulide producers real? To address these questions, we analyzed B. cereus strains isolated from a variety of sources, both cereulide-producing and cereulide-nonproducing strains, using MLST, and compared the phylogenetic allocation of the cereulide-producing strains with their serotypes.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Bacterial strains and serotyping. A collection of 52 strains of B. cereus, 25 cereulide-producing strains and 27 cereulide-nonproducing strains, was used in this study. The strains were collected from cases of emetic and diarrheal food poisoning, during regular food hygiene inspections, and from the environment (river, water, and soil sources). These strains were isolated between 1974 and 2005, mainly in Japan (Tables 1 and 2). Flagellar antigen serotyping was performed by the H antigen agglutination method described by Taylor and Gilbert (19).


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  		TABLE 1. Characteristics of cereulide-nonproducing strains of B. cereus

 

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  		TABLE 2. Characteristics of cereulide-producing strains of B. cereus

 
Toxin analyses. Cereulide production was tested by the vacuole response assay as described by Agata et al. (3). Enterotoxin production was tested by using a reversed passive latex agglutination B. cereus enterotoxin test kit (Oxoid, Basingstoke, United Kingdom) for HBL (hemolytic enterotoxin), using instructions provided by the manufacturer.

Molecular analyses. DNA was extracted according to the instructions for using a Promega Wizard genomic DNA purification kit (Promega K. K., Tokyo, Japan) after culture growth in 5 ml Luria-Bertani broth. MLST was performed as described on the B. cereus MLST Web site (http://pubmlst.org/bcereus). PCR amplification was performed on an Astec PC 802 thermal cycler (Astec, Fukuoka, Japan) as follows: 94°C for 5 min, 35 cycles of denaturation at 94°C for 1 min, annealing for 1 min (the recommended annealing temperature for each primer set was used), and extension at 72°C for 1 min, with final extension at 72°C for 7 min. The PCR amplicons were extracted following agarose gel electrophoresis using a QIAquick gel extraction kit (QIAGEN K.K., Tokyo, Japan). Sequencing was performed using a GenomeLab dye terminator cycle sequencing Quick Start kit (Beckman Coulter, Fullerton, CA) with the same primers used for the amplification, in both forward and reverse directions, and the reaction products were separated using a Beckman Coulter CEQ 2000 XL DNA analysis system (Beckman Coulter, Fullerton, CA). DNA sequences were assembled using Sequencher software (Gene Codes, Ann Arbor, MI).

Data analyses. The completed sequences were compared with those in the B. cereus MLST database. All new sequences were submitted to the database, and relevant data are available at http://pubmlst.org/bcereus. Concatenated sequences of all STs were downloaded from the database, and a neighbor-joining (NJ) tree was constructed using Clustal X (20) and visualized using Njplot (14). Split decomposition analysis was performed using the Web version of Splits Tree software on the MLST Web site (http://pubmlst.org) (12). eBURST analysis was performed using the Web tool at http://www.mlst.net/ (18).


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
All cereulide-producing strains of B. cereus were serotyped into one of three groups, H1, H3, and H12. In contrast, many cereulide-nonproducing strains could not be assigned to any of the 18 established serotypes described by Taylor and Gilbert (19), except for a few strains belonging to serotype H11. When tested for enterotoxin production, all cereulide-nonproducing strains were HBL positive, whereas all cereulide-producing strains were HBL negative (Table 1 and 2).

MLST revealed that cereulide-nonproducing strains were highly diverse, and 13 new STs were identified (data not shown). Four strains were allocated to ST 142. Another six strains were recovered in ST 24, and four of them were serotype H11, but not all H11 strains were ST 24. MLST analysis of cereulide-producing strains separated them into four STs (Table 3). Sequence types 165, 164, and 144 were new to the database. Two of these STs (ST 165 and 144) were single-nucleotide variants of their parent types, ST 26 and ST 164, respectively (data not shown). It is noteworthy that all strains allocated to ST 26 and its variant ST 165 were serotype H1. All strains in ST 144 were serotype H3, and all strains in ST 164 were serotype H12 (Table 3).


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  		TABLE 3. Changes in the allelic profiles of cereulide-producing strains of B. cereus

 
Phylogenetic analysis placed all emetic toxin-producing strains in Clade 1, as designated by Priest, et al. (16), clustered at two distinct loci: the previously known ST 26 with the newly identified variant ST 165 and the new ST 144/ST 164 locus (Fig. 1). The new cluster ST 144/ST 164 is located in the neighboring lineage to B. anthracis, distinct from the previously identified cluster at ST 26. Cereulide-nonproducing strains, from both the environment and from cases of food poisoning, were distributed in Clade 2, as designated by Priest, et al. (16), with only two exceptions. These strains had no significant relationships with each other and did not form a recognizable cluster (Fig. 2). We further subjected the group of emetic strains to eBURST analysis (implementation of the BURST [based upon related sequence types] algorithm) to look for phylogenetic links in their recent evolutionary history (18). However, the two clusters did not link even when the number of identical loci for group definition was decreased from the standard 6 out of 7 to 3 out of 7 (Fig. 3).


Figure 1
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  		FIG. 1. NJ phylogenetic tree of the B. cereus group population as represented in the B. cereus MLST database. The figure shows Clade 1, as designated by Priest, et al. (16). Arrows indicate the position of the STs of cereulide-producing strains analyzed in this study.

 

Figure 2
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  		FIG. 2. Split decomposition analysis of the group of cereulide-nonproducing strains of B. cereus analyzed in this study.

 

Figure 3
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  		FIG. 3. eBURST analysis of cereulide-producing strains of B. cereus analyzed in this study. FREQ, frequency (number of strains at each ST); SLV, single-locus variant; DLV, double-locus variant; TLV, triple-locus variant; SAT, satellites, more distantly related strains. Number of isolates, 24; number of STs, 4; number of resamplings for bootstrapping, 1,000; number of loci per isolate, 7; number of identical loci for group definition, 3; number of groups, 1. Group 1 number of isolates, 24; number of STs, 4; predicted founder, none.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Cereulide-nonproducing strains of B. cereus isolated from cases of food poisoning or the environment showed a high level of genetic diversity. Consistent with previously published results (16), they were distributed mainly in Clade 2, in close phylogenetic relationship to Bacillus thuringiensis. On the contrary, published reports have thus far assigned all analyzed cereulide-producing strains to a single phylogenetic cluster (9, 16). Based on MLST analysis, these strains have been assigned exclusively to ST 26 (16). Our study identified a strain with a new single mutation in ST 26, creating the new variant ST 165. We also recovered several cereulide-producing strains whose genetic sequences were at a location distinct from ST 26, in the lineage most closely related to the lineage populated by B. anthracis. Analysis by eBURST showed that this second cluster of cereulide-producing strains represents an independent group, with no detectable phylogenetic relationship to the previously reported cluster.

Previous studies have based their conclusions on strains from various European locations and on a few strains from North America (9, 16). To balance this, our collection of cereulide producers consisted predominantly of Japanese strains, in an effort to shed some light on genetic diversity in a geographically distinct region. Interestingly, all non-Japanese strains in our collection (total of three) fell into the newly identified cluster, together with a few Japanese strains. This distribution shows the rarity of ST 144/ST 164 strains and the predominance of ST 26 emetic strains worldwide.

Comparison of MLST analysis to flagellar typing provided a link between phylogenetic allocation and serotype. All strains of serotype H1 belonged to ST 26, while the newly identified cluster was formed exclusively of strains with serotypes H3 and H12. Our findings provide a link between phylogenetic location and flagellar serotype insofar as the clusters are distinguishable by their flagellar H serotype. It appears not only that cluster ST 26 (serotype H1) strains outnumber those of the new cluster ST 144/ST 164 (serotype H3/H12) but also that cluster ST 26 includes almost all food poisoning-associated strains. This correlates with the observations by Agata et al. that serotype H1 strains produce higher amounts of cereulide (1).

Recently published complete sequences of the cereulide synthetase gene cluster (7) and plasmid profiling work in emetic strains (11) as well as our unpublished data have shown that the operon responsible for cereulide production is situated on a large plasmid. Thorsen et al. recently reported that B. weihenstephanensis strains could produce cereulide (21). The fact that the capacity for cereulide production is shared by two phylogenetically distinct B. cereus clusters and by B. weihenstephanensis strains suggests that the plasmid can be subjected to lateral transfer.

It has been hypothesized that cereulide-producing strains, like B. anthracis, represent a recently emerged clone in the B. cereus population (9). So far, no substantial heterogeneity has been found among strains in the B. anthracis population (10, 13). The data presented here suggest that cereulide-producing strains are progressively diversifying.


   ACKNOWLEDGMENTS
 
This work was supported by a Grant-in-Aid for Scientific Research (17590388, 14370090 and 16590356) and by Research for the Future program from the Japanese Society for the Promotion of Science (JSPS-RFTF00L01411).

This publication made use of the Bacillus cereus Multi Locus Sequence Typing Website (http://pubmlst.org/bcereus/) developed by Keith Jolley and sited at the University of Oxford (Jolley et al. 2004; BMC Bioinformatics 5:86). The development of this site has been funded by the Wellcome Trust.


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of Bacteriology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan. Phone: 81-52-744 2106. Fax: 81-52-744 2107. E-mail: mohta{at}med.nagoya-u.ac.jp Back

{triangledown} Published ahead of print on 21 February 2007. Back


   REFERENCES
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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Journal of Clinical Microbiology, April 2007, p. 1274-1277, Vol. 45, No. 4
0095-1137/07/$08.00+0     doi:10.1128/JCM.02224-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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