1 Lehrstuhl für Mikrobielle Ökologie, Department für Grundlagen der Biowissenschaften, Technische Universität München, Weihenstephaner Berg 3, D-85354 Freising, Germany
2 Swedish Dairy Association, Scheelevaegen 18, 223 63 Lund, Sweden
3 Institut National de la Recherche Agronomique, UMR A408 Sécurité et Qualité des Produits d'Origine Végétale, INRA, Domaine Saint-Paul, Site Agroparc, F-84914 Avignon Cedex 9, France
4 Department of Pharmacology, Microbiology and Food Hygiene, The Norwegian School of Veterinary Science, Ullevalsveien 72, PO Box 8146, Dep., N-0033 Oslo, Norway
5 Dept for Applied Chemistry and Microbiology, College of Agriculture and Forestry at the University of Helsinki, Biocenter PO Box 56, Viikinkaari 9, FIN 00014 Helsinki University, Finland
6 Institute of Hygiene and Technology of Food of Animal Origin, Ludwig-Maximilians-Universität München, Veterinaerstr 13, D-80539 Munich, Germany
Correspondence
Siegfried Scherer
Siegfried.Scherer{at}wzw.tum.de
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the sequences of the internal gene fragments used for MLST and for the sporulation stage III AB genes reported in this paper are AY762151AY762213 and AY578317AY578349, respectively.
Full details of the strains used in this study and additional genetic relationship data are available as supplementary data with the online version of this paper at http://mic.sgmjournals.org.
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INTRODUCTION |
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B. cereus causes two types of food-poisoning syndromes: emesis and diarrhoea. The emetic syndrome is mainly characterized by vomiting a few hours after ingestion of contaminated food. In the diarrhoeal syndrome, symptoms appear 8 to 16 h after ingestion and include abdominal pain and diarrhoea. In general, both types of food-borne illness are relatively mild and usually do last not more than 24 h. Nevertheless, more severe cases have occasionally been reported, including one death after the ingestion of food contaminated with high amounts of emetic toxin and three deaths caused by a necrotic enterotoxin (Lund et al., 2000; Mahler et al., 1997
). Diarrhoeal poisoning is caused by heat-labile enterotoxins produced during vegetative growth of B. cereus in the small intestine (Granum, 1994
). At present, three different enterotoxins involved in food-poisoning outbreaks have been genetically characterized: two protein complexes, haemolysin BL (Hbl) (Beecher et al., 1995
) and non-haemolytic enterotoxin (Nhe) (Granum et al., 1999
), and the cytotoxin CytK (Lund et al., 2000
).
While enterotoxins are well characterized at the molecular and the expression level, far less is known about the emesis-causing toxin cereulide (for a review see Granum, 2001; Ehling-Schulz et al., 2004a
). Cereulide is a small, heat- and acid-stable cyclic dodecadepsipeptide (Agata et al., 1994
) and is synthesized by a non-ribosomal peptide synthetase, encoded by the ces genes (Ehling-Schulz et al., 2005
). It has been shown to cause cellular damage in animal models (Shinagawa et al., 1995
) and it has been reported that cereulide inhibits human natural killer cells and might therefore have an immunomodulating effect (Paananen et al., 2002
).
The trend towards Refrigerated Processed Foods of Extended Durability (REPFEDs) and the increasing percentage of elderly and immunocompromised people will raise the importance of B. cereus as an aetiological agent of food-borne illness. In addition to its food-poisoning potential, B. cereus has been shown to be responsible for wound and eye infections, as well as systemic infections and periodontitis, and was recently reported to be involved in an illness with symptoms resembling anthrax (Drobniewski, 1993; Hoffmaster et al., 2004
). While the genetic variability of environmental B. cereus isolates and isolates connected to periodontitis and other human such as eye infections, have been studied in some detail (Helgason et al., 2000b
), nothing is known about the population structure of food-poisoning B. cereus. Nevertheless, to understand epidemical processes, an assessment of the population structure of this food pathogen would be important. In addition, such a study, revealing the genetic diversity, may also contribute to a more appropriate detection and control of food-borne diseases caused by B. cereus.
In the present work we provide the first genotypic and phenotypic in-depth characterization of toxic B. cereus strains connected to food-borne illness. A comprehensive collection of clinical isolates, isolates from food remnants connected to food-poisoning outbreaks and isolates from food was compiled to assess the diversity of food-poisoning B. cereus. Emphasis was placed on emetic toxin producers because only limited information on emetic types is available.
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METHODS |
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Cloning and sequencing of RAPD amplicons.
DNA fragments of emetic and emetic-like strains were subcloned in Topo TA vector (Invitrogen) and sequenced as described previously (Ehling-Schulz et al., 2004b). The resulting sequences were searched against the sequenced genomes of B. cereus group strains and against the NCBI non-redundant protein database using BLAST (Altschul et al., 1990
).
Multilocus sequence typing (MLST) scheme.
Gene fragments from seven housekeeping genes [adk (encoding adenylate cyclase), ccpA (catabolic control protein A), ftsA (cell division protein), glpT (glycerol-3-phosphate permease), pyrE (orotate phosphoribosyl transferase), recF (DNA replication and repair protein) and sucC (succinyl coenzyme A synthetase)] were amplified from six emetic, one emetic-like and two non-emetic B. cereus strains, using the primers and amplification conditions described by Helgason et al. (2004). The sporulation stage III AB gene (spoIIIAB) was used as an additional genetic locus for sequence typing of food-poisoning and food strains (primer sequences used for amplification of the spoIIIAB gene are provided in Table 2
). PCR amplicons were purified using the QIAquick PCR purification kit (Qiagen) and directly sequenced (Sequiserve). Sequences were aligned and analysed using the CLUSTAL X software package (Thompson et al., 1997
) and compared to sequences available from databases. Nucleic acid sequence data for the above-mentioned housekeeping genes from B. cereus group strains, representing different sequence types (Helgason et al., 2004
), were retrieved from the GenBank database (accession nos AY387859AY388397) and used to assess the genetic relatedness among the sequenced emetic isolates. The software package TREECON was used for cluster analysis (Van de Peer & De Wachter, 1997
). Distances of sequences were calculated based on Kimura's two-parameter model (Kimura, 1980
) and UPGMA or the neighbour-joining method were used for inferring the tree topology.
Sequencing of 16S rDNA, 16S23S rDNA spacer region and 23S RNA.
Chromosomal DNA was extracted from single colonies using a ribolyser (Hybaid) according to the manufacturer's instructions. The primers used are listed in Table 2. The PCR protocol started with a denaturation step for 5 min at 95 °C, followed by 30 cycles of 20 s at 95 °C, 40 s at 55 °C, 2 min at 72 °C, and ended with a final elongation step at 72 °C for 5 min. PCR amplicons were purified, sequenced and analysed as described above.
Fourier transform infrared (FTIR) spectroscopy.
All isolates were grown as lawns of cells on tryptic soy agar (1·5 % CASO agar; Oxoid) at 25 °C for 24 h. The samples for FTIR spectroscopy were prepared as described by Oberreuter et al. (2002a) and spectra were recorded on an IFS 28/B spectrometer (Bruker Optics). Evaluation of IR spectral data was performed using OPUS software (version 3; Bruker Optics). The second derivations of the original spectra were calculated to enhance the resolution of superimposed bands and to minimize difficulties arising from unavoidable band shifts. Spectral distances which provide a measure of the similarity of spectra were used for cluster analysis.
Exoprotein profiling.
All isolates were cultivated in 50 ml CGY [2 % Casamino acids, 0·4 % glucose, 0·6 % yeast extract, 0·2 % (NH4)2SO4, 1·4 % KH2PO4, 0·1 % sodium citrate.H2O, 0·2 % MgSO4.7H2O, 0·05 % tryptophan] (Heinrichs et al., 1993) for 6 h at 32 °C. After centrifugation for 20 min at 8000 g (4 °C), the supernatants were 10-fold concentrated by using 80 % saturated ammonium sulphate precipitation. The precipitates were dissolved in 20 mM Tris/HCl, pH 7·6, and dialysed against the same buffer overnight at 4 °C. Fifteen microlitres of the dialysed sample was mixed with 5 µl 2x application buffer (125 mM Tris/HCl, pH 6·8, 20 % glycerol, 4 % SDS, 10 % 2-
-mercaptoethanol, 0·005 % bromophenol blue), boiled for 4 min and applied to a 10 % SDS-polyacrylamide gel. SDS-PAGE was carried out using a Bio-Rad Mini-Protean II Dual Slab Cell and the molecular mass of the proteins was estimated using SDS-PAGE low-range standards (Bio-Rad). The gels were stained using the Bio-Rad Silver Stain Plus kit.
Cluster analysis.
The RAPD and M13-patterns as well as the SDS protein profiles were analysed with GelCompar 4.1 (Applied Maths) using the Pearson correlation coefficients between the densitometric traces and the clustering method of Ward (1963). FTIR spectral analysis was carried out using the cluster analysis module of OPUS (Bruker Optics) and dendrograms were calculated on the basis of Ward's algorithm. Reproducibility of each method was tested by replicate samples from at least two different experiments.
Detection of emetic isolates and test for emetic toxin activity.
A recently developed PCR assay, targeting the cereulide synthetase (ces) gene (Ehling-Schulz et al., 2005), was applied to the entire test panel of strains to identify emetic B. cereus strains. Sequences of cereulide-synthetase-specific primers are provided in Table 2
; DNA extraction and PCR was performed as described previously (Ehling-Schulz et al., 2005
). Emetic toxin production was tested by a biological assay based on the loss of motility of sperm cells upon exposure to the emetic toxin produced by B. cereus (Jääskeläinen et al., 2003
) and a cell culture assay employing HEp-2 cells (Finlay et al., 1999
). Strains that carried the ces gene and were positive in the toxin assays were designated emetic strains.
Detection of enterotoxin genes and enterotoxin quantification.
For detection of the L2 component of Hbl (HblC) the BCET-RPLA B. cereus Enterotoxin Test Kit (Oxoid) was used. For detection of NheA the Tecra BDE kit (Tecra Diagnostics) was used. Both kits were used according to the instruction manuals on 10-fold concentrated culture supernatants. In addition, strains were tested for the presence of genes of the hbl operon and cytK using the primers listed in Table 2. PCR was performed according to Guinebretiere et al. (2002)
and Stenfors et al. (2002)
. Strains that were negative for hbl in the PCR assay were checked by Southern blotting, using hblD as probe according to Guinebretiere et al. (2002)
.
Starch hydrolysis, salicin fermentation and haemolysis.
The ability to hydrolyse starch and ferment salicin was tested according to Claus & Berkeley (1986) and Parry et al. (1983)
. Haemolysis was assessed by spot inoculation on tryptic soy agar (BBL) containing 5 % defibrinated bovine or ovine blood. The plates were incubated at 30 °C for 20 h. B. cereus F4810/72 (emetic) and ATCC 7064 (non-emetic) were always included as reference strains. The zone of haemolysis was classified as large (comparable to ATCC 7064), weak (compared to F4810/72) or absent.
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RESULTS |
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Allelic profiles of food-poisoning B. cereus
Based on seven housekeeping genes examined by using MLST loci (Helgason et al., 2004), seven emetic strains derived from different geographical locations (Finland, Germany, Japan, Netherlands, Sweden and UK) and sources (clinical, food remnants from food poisoning, and food) showed the same sequence type [see Fig. 3
and Fig. S1 (available as supplementary data with the online version of this paper at http://mic.sgmjournals.org), respectively]. An emetic-like strain shared the sequence type with emetic strains in six out of the seven loci.
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Exoprotein profiles were analysed by SDS-PAGE (Fig. 1). The reproducibility was lower than that achieved with the other typing methods. Nevertheless, it is clear that most of the emetic isolates clustered together, separate from the other isolates, in subgroups A1 and A2 (Table 3
). Interestingly, in cluster D only isolates from food-poisoning outbreaks were observed, whereas cluster E was dominated by food isolates (Fig. 2
).
Toxin profiles
The entire set of strains (Table 1) was tested for the presence of toxin genes and production of toxins that have been ascribed to be involved in diarrhoea and emetic food-poisoning outbreaks. Twenty-two isolates derived from food-poisoning cases and two (SDA GR177 and SDA GR177a) out of the 30 food isolates tested carried the ces (cereulide synthetase) gene and produced the emetic toxin cereulide as determined by the boar spermatozoa assay and cell culture assay. None of these emetic isolates produced Hbl, nor did any of them carry the hbl genes (Table 3
), whereas Hbl production was frequently observed among isolates derived from diarrhoeal-type food poisoning and from different food sources as well (Table 4
).
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Biochemical tests
Emetic strains showed only weak or no haemolysis (Fig. 5) on blood agar, were unable to degrade starch and were negative for salicin fermentation (Table 3
), while the majority of isolates from diarrhoeal outbreaks and foods showed haemolysis. Many of these strains displayed signs of discontinuous haemolysis due to Hbl, in agreement with the presence of the hbl genes. The inability to degrade starch was also found frequently among non-emetic strains. However, all strains producing Hbl were starch-positive (Table 4
), while 71 % of starch-negative strains showed only weak haemolysis. Therefore, besides production of cereulide, so far no phenotypic characteristics are known which are unique for emetic B. cereus.
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DISCUSSION |
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Haemolytic-enterotoxin-producing B. cereus strains are polymorphic
Three to four major branches were observed in all tree topologies derived from genotypic and phenotypic data of food-poisoning B. cereus, which is in agreement with results of multilocus enzyme electrophoresis (MLEE) and MLST that revealed three major groups for the total B. cereus group (Helgason et al., 2004). Our results indicate a high degree of heterogeneity among enterotoxic B. cereus isolates derived from diarrhoeal outbreaks and food. Hbl formation, for instance, was observed in 53 % of the food isolates belonging to different clusters, which is in accordance with results reported previously. Prüß et al. (1999)
showed that the enterotoxin Hbl is broadly distributed among B. cereus group strains (about 50 %) and that it relates neither to a certain species nor to a specific environment. In general, cluster analysis revealed a broader distribution for isolates from food environments than for clinical isolates related to food poisoning (Fig. 2
). Helgason et al. (2000b)
used MLEE to study the genetic diversity of dairy and soil isolates of B. cereus and B. thuringiensis in comparison to the genetic diversity of clinical isolates obtained from periodontitis and other human infections. As observed in our study, their results revealed a higher degree of heterogeneity among food and environmental strains, than observed among clinical isolates. However, no data on toxin profiles of the strains analysed were provided by the latter study.
Emetic B. cereus strains form a single monomorphic cluster
The diarrhoeal strains have been collected mainly from Europe. Nevertheless, they displayed a high degree of diversity, while all emetic strains, including an isolate from Japan, showed only a low genetic variability in eight chromosomal loci as well as in genotypic characteristics as shown by M13-PCR and RAPD. The biochemical tests and phenetic typing methods FTIR and exoprotein profiling, as well as the analysis of potential virulence factors like enterotoxin genes, provided further evidence for a very close relationship among isolates belonging to the emetic type of B. cereus. It has been previously reported, based on a few emetic-toxin-producing B. cereus isolates, that these isolates possess similar ribotypes (Pirttijärvi et al., 1999), which is supported by the observation that rDNA sequences of emetic strains are identical (16S, ITS, 5' region of 23S rDNA). We therefore suggest an emetic type of B. cereus which is restricted to a genetic lineage of strains sharing various phenotypic and genotypic characteristics, such as the inability to hydrolyse starch and salicin, having only weak haemolytic activity or no haemolytic activity at all and the absence of the hbl gene that is otherwise commonly found in B. cereus.
Some strains cluster closely to emetic isolates, but do not produce the emetic toxin cereulide. It may be speculated that these emetic-like isolates have lost their capability for toxin production. This is supported by the observation that the cell-surface protein is highly conserved among emetic and emetic-like strains (Table 5), while the ces gene responsible for non-ribosomal synthesis of cereulide (Ehling-Schulz et al., 2005
) is restricted to emetic-toxin-producing strains and is not found in emetic-like strains (see Tables 3 and 4
).
Epidemic population structure of B. cereus?
The population structure of pathogenic bacteria varies over a wide range, from strictly clonal to effectively panmictic (Suerbaum et al., 1998; Maynard-Smith et al., 2000
; Dykhuizen & Baranton, 2001
; Feil & Spratt, 2001
; Feil et al., 2003
). It has been reported that gene exchange between B. cereus and B. thuringiensis does occur and that sufficient recombination in B. cereus populations is observed to consider this species as being panmictic (Vilas-Boas et al., 2002
). The high degree of molecular diversity among haemolytic enterotoxic isolates connected to food poisoning may also point towards a more panmictic population structure of isolates connected to the diarrhoeal syndrome. The data from the MLST analysis add to this argument. The individual tree topologies derived from different loci are not identical [Helgason et al., 2004
; this work, see Fig. S1 (available as supplementary data with the online version of this paper at http://mic.sgmjournals.org)]. Such contradictions in cluster formation have been interpreted as being the result of recombination (Dykhuizen & Green, 1991
; Maynard-Smith et al., 2000
). On the other hand, Priest et al. (2004)
have used an alternative set of genes for an MLST analysis of 105 strains of all B. cereus group species. From their data, they inferred a generally clonal population structure. Whether recombination in B. cereus is sufficiently frequent to generate linkage equilibrium (i.e. complete panmixis) or whether the population structure is weakly clonal (Maynard-Smith et al., 2000
) needs further investigation.
The data presented in this work demonstrate a remarkably homogeneous virulent emetic clone of B. cereus. Whether B. cereus, has an epidemic population structure which is characterized by a rather panmictic background population recombining with a relatively high frequency, superimposed by strains with closely related genotypes forming clonal complexes (Maynard-Smith et al., 2000; see also Feil & Spratt, 2001
) is not yet clear.
Why the emetic clonal complex has evolved within B. cereus is currently unknown. Future research to resolve this question may be geared into two directions: first, towards finding potential differences in recombinatorial capacity of emetic B. cereus compared to diarrhoeal strains and, second, towards the possibility that the production of emetic toxin has been acquired relatively recently in the evolution of this lineage, providing a selective advantage. The latter hypothesis is supported by the very low molecular diversity of the genetic locus that is responsible for cereulide synthesis. Sequencing of 2·2 kb from the ces genes from 12 emetic B. cereus strains revealed only a single neutral nucleotide difference at a wobble position (Ehling-Schulz et al., 2005; this work). Such a lack of molecular polymorphism was also reported for B. anthracis. Comparative analysis of the protective antigen gene sequence revealed 5 nt differences across 2·5 kb (Price et al., 1999
) and a clonal population structure has been suggested for B. anthracis based on its lack of molecular polymorphism in its genome as well as in its virulence plasmids pXO1 and pXO2 (Keim et al., 1997
; Hill et al., 2004
).
In conclusion, it is tempting to speculate that the emetic clonal complex of B. cereus emerged recently through the acquisition of key virulence factors such as cereulide synthetase, which is absent in non-emetic strains (Ehling-Schulz, 2005). Due to its recent formation, the influence of recombination and point mutation on this clone may still be low.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. (1990). Basic local alignment search tool. J Mol Biol 215, 403410.[CrossRef][Medline]
Beecher, D. J., Schoeni, J. L. & Wong, A. C. L. (1995). Enterotoxic activity of hemolysin BL from Bacillus cereus. Infect Immun 63, 44234428.[Abstract]
Claus, D. & Berkeley, R. C. W. (1986). Genus Bacillus Cohn 1872, 174AL. In Bergey's Manual of Systematic Bacteriology, vol. 2, pp. 11051139. Edited by P. H. A. Sneath, N. S. Mair, M. E. Sharpe & J. G. Holt. Baltimore, MD: Williams & Wilkins.
Daffonchio, D., Borin, S., Frova, G., Gallo, R., Mori, E., Fani, R. & Sorlini, C. (1999). A randomly amplified polymorphic DNA marker specific for the Bacillus cereus group is diagnostic for Bacillus anthracis. Appl Environ Microbiol 65, 12981303.
Drobniewski, F. A. (1993). Bacillus cereus and related species. Clin Microbiol Rev 6, 324338.[Abstract]
Dykhuizen, D. E. & Baranton, G. (2001). The implications of a low rate of horizontal transfer in Borrelia. Trends Microbiol 9, 344350.[CrossRef][Medline]
Dykhuizen, D. E. & Green, L. (1991). Recombination in Escherichia coli and the definition of biological species. J Bacteriol 173, 72577268.[Medline]
Ehling-Schulz, M., Fricker, M. & Scherer, S. (2004a). Bacillus cereus, the causative agent of an emetic type of foodborne illness. Mol Nutr Food Res 48, 479487.[CrossRef][Medline]
Ehling-Schulz, M., Fricker, M. & Scherer, S. (2004b). Identification of emetic toxin producing Bacillus cereus strains by a novel molecular assay. FEMS Microbiol Lett 232, 189195.[CrossRef][Medline]
Ehling-Schulz, M., Vukov, N., Schulz, A., Shaheen, R., Andersson, M., Märtlbauer, E. & Scherer, S. (2005). Identification and partial characterization of the nonribosomal peptide synthase gene responsible for cereulide production in emetic Bacillus cereus. Appl Environ Microbiol 71 (in press).
Feil, E. J. & Spratt, B. G. (2001). Recombination and the population structures of bacterial pathogens. Annu Rev Microbiol 55, 561590.[CrossRef][Medline]
Feil, E. J., Cooper, J. E., Grundmann, H. & 9 other authors (2003). How clonal is Staphylococcus aureus? J Bacteriol 185, 33073316.
Finlay, W. J., Logan, N. A. & Sutherland, A. D. (1999). Semiautomated metabolic staining assay for Bacillus cereus emetic toxin. Appl Environ Microbiol 65, 18111812.
Gordon, R. E., Haynes, W. C. & Pang, C. H.-N. (1973). The genus Bacillus. Washington, DC: US Department of Agriculture.
Granum, P. E. (1994). Bacillus cereus and its toxins. J Appl Bacteriol Symp Suppl 23, 61S66S.
Granum, P. E. (2001). Bacillus cereus. In Food Microbiology: Fundamentals and Frontiers, pp. 373381. Edited by M. P. Doyle & others. Washington, DC: American Society for Microbiology.
Granum, P. E., O'Sullivan, K. & Lund, T. (1999). The sequence of a non-hemolytic enterotoxin operon from Bacillus cereus. FEMS Microbiol Lett 177, 225229.[CrossRef][Medline]
Guinebretiere, M. H., Broussolle, V. & Nguyen-The, C. (2002). Enterotoxigenic profiles of food-poisoning and food-borne Bacillus cereus strains. J Clin Microbiol 40, 30533056.
Gürtler, V. & Stanisich, V. A. (1996). New approaches to typing and identification of bacteria using the 16S23S rDNA spacer region. Microbiology 142, 316.[Medline]
Heinrichs, J. H., Beecher, D. J., Macmillan, J. D. & Zilinskas, B. A. (1993). Molecular cloning and characterization of the hblA gene encoding the B component of hemolysin BL from Bacillus cereus. J Bacteriol 175, 67606766.[Abstract]
Helgason, E., Okstad, O. A., Caugant, D. A., Johansen, H. A., Fouet, A., Mock, M., Hegna, I. & Kolstø, A. B. (2000a). Bacillus anthracis, Bacillus cereus and Bacillus thuringiensis one species on the basis of genetic evidence. Appl Environ Microbiol 66, 26272630.
Helgason, E., Caugant, D. A., Olsen, I. & Kolstø, A. B. (2000b). Genetic structure of population of Bacillus cereus and Bacillus thuringiensis associated with periodontitis and other human infections. J Clin Microbiol 38, 16151622.
Helgason, E., Tourasse, N. J., Meisal, R., Caugant, D. A. & Kolstø, A. B. (2004). Multilocus sequence typing scheme for bacteria of the Bacillus cereus group. Appl Environ Microbiol 70, 191201.
Henderson, I., Duggleby, C. J. & Turnbull, P. C. (1994). Differentiation of Bacillus anthracis from other Bacillus cereus group bacteria with the PCR. Int J Syst Bacteriol 44, 99105.[Abstract]
Hill, K. K., Ticknor, L. O., Okinaka, R. T. & 13 other authors (2004). Fluorescent amplified fragment length polymorphism analysis of Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis isolates. Appl Environ Microbiol 70, 10681080.
Hoffmaster, A. R., Ravel, J., Rasko, D. A. & 19 other authors (2004). Identification of anthrax toxin genes in a Bacillus cereus associated with an illness resembling inhalation anthrax. Proc Natl Acad Sci U S A 101, 84498454.
Jääskeläinen, E. I., Häggblom, M. M., Andersson, M. A., Vanne, L. & Salkinoja-Salonen, M. (2003). Potential of Bacillus cereus for producing an emetic toxin in bakery products: quantitive analysis by chemical and biological methods. J Food Protec 66, 10471054.
Keim, P., Kalif, A., Schupp, J. & 7 other authors (1997). Molecular evolution and diversity in Bacillus anthracis as detected by amplified fragment length polymorphism markers. J Bacteriol 179, 818824.[Abstract]
Kimura, M. (1980). A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16, 111120.[Medline]
Lechner, S., Mayr, R., Francis, K. P., Prüß, B. M., Kaplan, T., Wießner-Gunkel, E. G., Stewart, G. S. & Scherer, S. (1998). Bacillus weihenstephanensis sp. nov. is a new psychrotolerant species of the Bacillus cereus group. Int J Syst Bacteriol 48, 13731382.
Levier, D., Hirst, D., Holt, C. & Williams, A. G. (1997). Effect of sampling procedure and strain variation in Listeria monocytogenes on the discrimination of species in the genus Listeria by Fourier transform infrared spectroscopy and canonical variates analysis. FEMS Microbiol Lett 147, 4550.[CrossRef][Medline]
Lund, T., De Buyser, M. L. & Granum, P. E. (2000). A new cytotoxin from Bacillus cereus that may cause necrotic enteritis. Mol Microbiol 38, 254261.[CrossRef][Medline]
Mahler, H., Pasi, A., Kramer, J. M., Schulte, P., Scoging, A. C., Bär, W. & Krähenbühl, S. (1997). Fulminant liver failure in association with the emetic toxin of Bacillus cereus. N Engl J Med 336, 11421148.
Maoz, A., Mayr, R. & Scherer, S. (2003). Temporal stability and biodiversity of two complex, anti-listerial cheese ripening microbial consortia. Appl Environ Microbiol 69, 40124018.
Maynard-Smith, J., Feil, E. J. & Smith, N. H. (2000). Population structure and evolutionary dynamics of pathogenic bacteria. BioEssays 22, 11151122.[CrossRef][Medline]
Nilsson, J., Svensson, B., Ekelund, K. & Christiansson, A. (1998). A RAPD-PCR method for large-scale typing of Bacillus cereus. Lett Appl Microbiol 27, 168172.[CrossRef][Medline]
Oberreuter, H., Seiler, H. & Scherer, S. (2002a). Identification of coryneform bacteria and related taxa by Fourier-transformed infrared (FT-IR) spectroscopy. Int J Syst Evol Microbiol 52, 91100.
Oberreuter, H., Charzinski, J. & Scherer, S. (2002b). Intraspecific diversity of Brevibacterium linens, Corynebacterium glutamicum and Rhodococcus erythropolis based on partial 16S rDNA sequence analysis and Fourier-transform infrared (FT-IR) spectroscopy. Microbiology 148, 15231532.[Medline]
Paananen, A., Mikkola, R., Sareneva, T., Matikainen, S., Hess, M., Andersson, M., Julkunen, I., Salkinoja-Salonen, M. S. & Timonen, T. (2002). Inhibition of human natural killer cell activity by cereulide, an emetic toxin from Bacillus cereus. Clin Exp Immunol 129, 420428.[CrossRef][Medline]
Parry, J. M., Turnbull, P. C. B. & Gibson, J. R. (1983). A Colour Atlas of Bacillus Species. Wolfe Medical Publications Ltd.
Pirttijärvi, T. S., Andersson, M. A., Scoging, A. C. & Salkinoja-Salonen, M. S. (1999). Evaluation of methods for recognizing strains of the Bacillus cereus group with food poisoning potential among industrial and environmental contaminants. Syst Appl Microbiol 22, 133144.[Medline]
Price, L. B., High-Jones, M. E., Jackson, P. & Keim, P. (1999). Natural genetic diversity in the protective antigen gene of Bacillus anthracis. J Bacteriol 181, 23582362.
Priest, F. G., Goodfellow, M. & Todd, C. (1988). A frequency matrix for probabilistical identification of some bacilli. J Gen Microbiol 134, 18471882.[Medline]
Priest, F. G., Barker, M., Baillie, L. W. J., Holmes, E. C. & Maiden, M. J. C. (2004). Population structure and evolution of the Bacillus cereus group. J Bacteriol 186, 79597970.
Prüß, B. M., Dietrich, R., Nibler, B., Märtelbauer, E. & Scherer, S. (1999). The hemolytical enterotoxin HBL is broadly distributed among species of the Bacillus cereus group. Appl Environ Bacteriol 65, 54365442.
Shinagawa, K., Konuma, H., Sekita, H. & Sugii, S. (1995). Emesis of rhesus monkeys induced by intragastric administration with the HEp-2 vacuolation factor (cereulide) produced by Bacillus cereus. FEMS Microbiol Lett 130, 8790.[CrossRef][Medline]
Stackebrandt, E. & Liesack, W. (1992). The potential of rDNA in identification and diagnostics. In Nonradioactive Labeling and Detection of Biomolecules, pp. 232239. Edited by C. Kessler. Berlin: Springer.
Stenfors, L. P., Mayr, R., Scherer, S. & Granum, P. E. (2002). Pathogenic potential of fifty Bacillus weihenstephanensis strains. FEMS Microbiol Lett 215, 4751.[CrossRef][Medline]
Suerbaum, S., Maynard-Smith, J. M., Bapumia, K., Morelli, G., Smith, N. H., Kunstmann, E., Dyrek, I. & Achtman, M. (1998). Free recombination within Helicobacter pylori. Proc Natl Acad Sci U S A 95, 1261912624.
Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & Higgins, D. G. (1997). The CLUSTAL X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 24, 48764882.[CrossRef]
Tindall, B. J., Brambilla, E., Steffen, M., Neumann, R., Pukall, R., Kroppenstedt, R. M. & Stackebrandt, E. (2000). Cultivatable microbial biodiversity: gnawing at the Gordian knot. Environ Microbiol 2, 310318.[CrossRef][Medline]
Turnbull, P. C. B. & Kramer, J. M. (1991). Bacillus. In Manual of Clinical Microbiology, 5th edn, pp. 296303. Edited by A. Balows, W. J. Hausler, K. L. Hermann, H. D. Isenberg & H. J. Shadomy. Washington, DC: American Society for Microbiology.
Van de Peer, Y. & De Wachter, R. (1997). Construction of evolutionary distance trees with TREECON for Windows: accounting for variation in nucleotide substitution rate among sites. Comput Appl Biosci 13, 227230.[Abstract]
Van Leeuwen, W., Sijmons, M., Sluijs, J., Verbrugh, H. & van Belkum, A. (1996). On the nature and use of randomly amplified DNA from Staphylococcus aureus. J Clin Microbiol 34, 27702777.[Abstract]
Vilas-Boas, G., Sanchis, V., Lereclus, D., Lemos, M. V. F. & Bourguet, D. (2002). Genetic differences between sympatric populations of Bacillus cereus and Bacillus thuringiensis. Appl Environ Microbiol 68, 14141424.
Ward, J. H. (1963). Hierarchical grouping to optimize an objective function. J Am Stat Assoc 58, 236244.
Received 8 September 2004;
revised 28 September 2004;
accepted 14 October 2004.
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