Emetic toxin formation of Bacillus cereus is restricted to a single evolutionary lineage of closely related strains

Monika Ehling-Schulz1, Birgitta Svensson2, Marie-Helene Guinebretiere3, Toril Lindbäck4, Maria Andersson5, Anja Schulz6, Martina Fricker1, Anders Christiansson2, Per Einar Granum4, Erwin Märtlbauer6, Christophe Nguyen-The3, Mirja Salkinoja-Salonen5 and Siegfried Scherer1

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


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
An in-depth polyphasic approach was applied to study the population structure of the human pathogen Bacillus cereus. To assess the intraspecific biodiversity of this species, which is the causative agent of gastrointestinal diseases, a total of 90 isolates from diverse geographical origin were studied by genetic [M13-PCR, random amplification of polymorphic DNA (RAPD), multilocus sequence typing (MLST)] and phenetic [Fourier transform Infrared (FTIR), protein profiling, biochemical assays] methods. The strain set included clinical strains, isolates from food remnants connected to outbreaks, as well as isolates from diverse food environments with a well documented strain history. The phenotypic and genotypic analysis of the compiled panel of strains illustrated a considerable diversity among B. cereus connected to diarrhoeal syndrome and other non-emetic food strains, but a very low diversity among emetic isolates. Using all typing methods, cluster analysis revealed a single, distinct cluster of emetic B. cereus strains. The isolates belonging to this cluster were neither able to degrade starch nor could they ferment salicin; they did not possess the genes encoding haemolysin BL (Hbl) and showed only weak or no haemolysis. In contrast, haemolytic-enterotoxin-producing B. cereus strains showed a high degree of heterogeneity and were scattered over different clusters when different typing methods were applied. These data provide evidence for a clonal population structure of cereulide-producing emetic B. cereus and indicate that emetic strains represent a highly clonal complex within a potentially panmictic or weakly clonal background population structure of the species. It may have originated only recently through acquisition of specific virulence factors such as the cereulide synthetase gene.


Abbreviations: FTIR, Fourier transform Infrared; Hbl, haemolysin BL; MLST, multilocus sequence typing; Nhe, non-haemolytic enterotoxin; RAPD, random amplification of polymorphic DNA

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 AY762151–AY762213 and AY578317–AY578349, 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.


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacillus cereus is a Gram-positive endospore-forming opportunistic human pathogen of the B. cereus species group. This group comprises the six species B. cereus, Bacillus mycoides, Bacillus pseudomycoides, Bacillus thuringiensis, Bacillus weihenstephanensis and Bacillus anthracis (Gordon et al., 1973; Lechner et al., 1998; Priest et al., 1988; Turnbull & Kramer 1991). While a high degree of diversity concerning the virulence factors is found, a close genetic relationship has been observed between all species of the B. cereus group (Helgason et al., 2000a). It was therefore suggested that the entire group represents a single species.

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.


   METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Strains.
A collection of 90 B. cereus isolates was compiled for this study. The strain set includes clinical isolates and isolates from food remnants connected to food-borne outbreaks, as well as isolates from diverse food stuffs and environment. Details on the origin of strains are given in Table 1 and further information on strains is provided in Table S1 (available as supplementary data with the online version of this paper at http://mic.sgmjournals.org).


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Table 1. Origin of B. cereus strains

 
M13-PCR typing.
M13-PCR typing is based on random PCR amplification of DNA fragments using an Escherichia coli phage M13-based primer. It was adapted from Henderson et al. (1994) using the sequence-specific primer PM13 (for primer sequences see Table 2). DNA was prepared as described previously (Guinebretiere et al., 2002). Each amplification started with a denaturation step of 3 min at 94 °C followed by 35 cycles of 1 min at 94 °C, 1 min at 40 °C, 8 min at 65 °C, and ended with a final elongation step of 16 min at 65 °C. PCR products were separated in a 2 % agarose gel, stained with ethidium bromide and digitized using a gel imager (Bioblock).


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Table 2. PCR primers used in this study

 
Random amplification of polymorphic DNA (RAPD).
RAPD-PCR was performed as described by Nilsson et al. (1998). The sequences of the arbitrary primers are provided in Table 2. The software GelCompar 4.1 (Applied Maths) enabled data from different sampling events to be combined in dendrograms, and allowed the comparison between RAPD-fingerprints of B. cereus isolates sampled over long time periods. Clusters containing identical RAPD patterns were confirmed by visual comparison of the banding patterns and by RAPD using an alternative primer (Table 2). Isolates with known RAPD profiles were included in each experiment as standard and positive control to check for the quality of PCR reactions (for details see Nilsson et al., 1998).

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 AY387859–AY388397) 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, 16S–23S 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-{beta}-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.


   RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Genotyping of food-poisoning B. cereus
An M13 coliphage-based primer (M13-PCR) and two additional arbitrary primers (RAPD-PCR) were used for genomic fingerprinting of 90 B. cereus isolates derived from food-borne outbreaks and food environments (representative profiles are shown in Fig. 1). Replica samples from independent experiments clustered at a 90 (M13-PCR) to 95 % (RAPD) level in the dendrogram calculated on the basis of Ward's algorithm (data not shown). With both typing methods, three to four major clusters are apparent that may represent distinct branches of B. cereus. The most prominent separation in the dendrograms is the split between emetic B. cereus isolates and enterotoxic isolates connected to the diarrhoeal syndrome. A third major group was formed by food isolates and psychrotolerant B. weihenstephanensis strains (Fig. 2).



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Fig. 1. Characteristic profiles of RAPD, M13 and SDS-PAGE analyses of emetic (lanes 1–3) and non-emetic (lanes 4–6) B. cereus strains. Lanes: 1, F4810/72; 2, NC7401; 3, RIVM-BC51; 4, F4430/73; 5, WSBC10030; 6, INRA SZ; non-emetic strains belong to different clusters (compare Tables 3 and 4).

 


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Fig. 2. Schematic dendrograms depicting the similarities between 90 B. cereus isolates from food-poisoning outbreaks and diverse food environments typed by M13-PCR (a), RAPD (b), FTIR spectroscopy (c) and exoprotein profiling (d). A1–A4 represent emetic strains or ‘emetic-like’ strains (grouped by grey shading). Black symbols originate from food-poisoning isolates; white symbols originate from food not associated with food poisoning. Circles, emetic toxin; triangles, Hbl toxin; squares, CytK toxin gene; diamonds, Nhe toxin or non-toxic.

 
According to M13-PCR, 34 isolates with identical or very similar M13-PCR patterns belong to group A, while 37 isolates with a similar or identical RAPD pattern cluster to the corresponding RAPD group A (Table 3). This group contained strains that have been isolated from emetic poisoning cases and a few strains that have been isolated from different food sources. All of these isolates were starch- and salicin-negative, exhibited weak or no haemolysis on blood agar and were hbl-negative. However, not all isolates in this group produced detectable amounts of emetic toxin or gave a signal in the emetic-strain-specific PCR. These were named ‘emetic-like’ isolates. RAPD patterns of the emetic and emetic-like isolates showed two bands (approx. 650 and 260 bp) that were not present in other isolates (representative RAPD patterns are shown in Fig. 1). Groups B and C contained the majority of enterotoxic strains connected to diarrhoeal syndrome (75 % according to M13-PCR and 72 % according to RAPD fingerprinting) while Group D, derived from M13-PCR as well as from RAPD, was dominated by food isolates (Fig. 2).


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Table 3. Characteristics of emetic and ‘emetic-like’ B. cereus isolates from food environments and clinical strains connected to food poisoning

 
Identification of RAPD markers for the emetic type of B. cereus
The emetic cluster (cluster A)-specific 650 and 250 bp RAPD bands were subcloned. Sequence analysis revealed a high homology of both 650 and 250 bp inserts to a cell-surface protein from B. cereus group members (data not shown). A genome BLAST search of the sequenced B. cereus group strains was performed, the corresponding cell surface gene sequences were retrieved and sequences were aligned using CLUSTAL X (Thompson et al., 1997). Oligonucleotide primers located in conserved regions upstream and downstream of the sequenced RAPD fragments (Table 2) were used to expand the nucleic acid sequence information obtained from the RAPD fragments and to determine potential RAPD primer-binding sites. This analysis revealed that molecular polymorphisms at RAPD primer-binding sites are responsible for the specific amplification of the 650 and 250 bp DNA fragments in emetic and emetic-like B. cereus strains (see Table 5) while the corresponding cell surface protein is not specific for emetic B. cereus.


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Table 5. Specific emetic B. cereus RAPD primer-binding site in a putative cell-surface gene (for details see text)

 
Analysis of rDNA sequences
Almost complete 16S rDNA data of seven emetic B. cereus strains from five different geographical locations (Finland, Germany, Japan, Sweden and UK) and partial 16S rRNA sequences from four non-emetic strains and the B. mycoides and B. thuringiensis type strains as well as from two B. anthracis strains and two B. weihenstephanensis strains were determined and compared to sequences available from databases. In general, almost no differences among the mesophilic members of the B. cereus group were found. In addition, the spacer region between the 16S and 23S rDNA genes and partial 23S rDNA were sequenced. The seven emetic strains sequenced displayed identical ITS sequences, which differed by a single nucleotide from sequences of non-emetic mesophilic B. cereus and B. anthracis, by 2 nt from B. thuringiensis, but by 11 nt from psychrotolerant B. weihenstephanensis and B. mycoides (data not shown). Emetic strains showed a heterologous base at position 19 (70 % A and 30 % T), the emetic-like strain showed a mixture of three nucleotides at that position (70 % A, 15 % T and 15 % C) while a C was observed in all B. cereus group strains without an emetic toxin profile.

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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Fig. 3. Genetic relationship among emetic B. cereus isolates inferred from concatenated housekeeping gene sequences according to Helgason et al. (2004). The tree was constructed with TREECON (Van de Peer & De Wachter, 1997) using the neighbour-joining method. All bootstrap values >80 % (1000 repeats) are shown next to the nodes. Sequences designated AHxxx were obtained from Helgason et al. (2004). For details on emetic strains, see Table 3 and Table S1 (available as supplementary data with the online version of this paper at http://mic.sgmjournals.org), respectively.

 
The sporulation stage III AB gene (spoIIIAB) was used as an additional chromosomal genetic locus to assess the genetic relatedness among food-poisoning isolates. The tree topology calculated from 32 B. cereus sequences branches in three to four major clusters (Fig. 4) as was observed with the M13 and RAPD methods (Fig. 2). In accordance with the MLST data, all emetic and emetic-like isolates cluster together (Fig. 4).



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Fig. 4. Genetic relationship observed among food and food-poisoning B. cereus strains using the spoIIIAB gene as chromosomal genetic marker. Distances of sequences were calculated based on Kimura's two-parameter model (Kimura, 1980) and UPGMA was used for inferring the tree topology. Bootstrap values (100 bootstrap replicates) are shown at the major nodes. Details on strain characteristics are provided in Tables 3 and 4. # indicates emetic-like strains.

 
Phenetic typing of food-poisoning B. cereus
FTIR spectra and exoprotein profiles were recorded from all 90 B. cereus isolates included in the test panel. Analysis of the second derivation of the FTIR spectra (data not shown) revealed five distinct clusters, designated A–E, as observed for exoprotein profiling. Spectra of all strains were recorded two times independently to test the reproducibility (data not shown). Cluster A was restricted to emetic-toxin-producing isolates or emetic-like isolates, whereas enterotoxin-producing strains were scattered throughout the other four clusters (Fig. 2). Clusters B, C and D were dominated by food-poisoning strains while most of the food isolates were found in cluster E.

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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Table 4. Characteristics of non-emetic B. cereus isolates from food environments and clinical strains connected to food poisoning

 
All isolates, except strain NHV 391-98, carried the nhe genes that encode Nhe. However, in only 71 % of the emetic-toxin-producing strains was nhe gene expression observed using immunological detection of NheA (Tecra). All Hbl-positive strains (Oxoid) were also positive in the immunoassay detecting NheA. cytK encodes a novel cytotoxin that was originally isolated from strain NVH 391-98 (Lund et al., 2000) and was detected in 63 % of the Hbl-positive strains but only in 8 % (two isolates, probably representing one strain) of the emetic strains.

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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Fig. 5. Typical haemolysis zones on (a) a bovine blood agar plate and (b) a sheep blood agar plate of emetic and non-emetic B. cereus strains. For strain designation, see Table 1.

 
Comparison of genetic and phenetic typing methods
In general, a strong correlation between the clustering of emetic and emetic-like strains (cluster A), according to genetic and phenetic methods, was observed, while only a low correlation between the different methods was found for enterotoxic strains isolated from food-borne outbreaks (see Tables 3 and 4, respectively). Food isolates showed some tendency to cluster separately from food-poisoning isolates (Fig. 2). FTIR and protein profiles confirmed the molecular data with only a few exceptions, mainly concerning isolates with an emetic profile that were negative for emetic toxin production, mesophilic, did not carry the ces genes nor the hbl genes, expressed no or only weak haemolysis on blood agar, were unable to metabolize starch and did not show salicin fermentation. We have associated the phenotype emetic-like to these strains.


   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Evaluation of typing methods to screen for emetic B. cereus
This study showed that FTIR and RAPD may be useful methods for rapid screening of potential emesis-causing B. cereus isolates, while M13-PCR and protein profiling are rather difficult to perform on a routine basis. FTIR is a fast technique that has already been demonstrated to be suitable for classification and identification of micro-organisms at the species and subspecies level (e.g. Levier et al., 1997; Oberreuter et al., 2002b; Maoz et al., 2003), as well as for rapid screening of environmental isolates (Tindall et al., 2000). The recently developed MLST scheme for B. cereus (Helgason et al., 2004) is suitable to study the genetic relationship among emetic food-poisoning isolates. However, since this method requires extensive sequencing effort, its applicability for routine screening and diagnostic purposes is limited. On the other hand, the identified chromosomal RAPD markers which are restricted to emetic and emetic-like strains might provide an interesting alternative for the rapid characterization of isolates belonging to the emetic subtype of B. cereus. RAPD fingerprinting has been proposed as a tool for generating taxon-specific markers with different specificities (e.g. Van Leeuwen et al., 1996) and has been successfully applied to discriminate B. anthracis from other B. cereus group members (Daffonchio et al., 1999).

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.


   ACKNOWLEDGEMENTS
 
This work was supported by the European Commission (QLK1-CT-2001-00854). We thank Katrin Buntin, Kerstin Ekelund, Amanda Montan and Ranad Shaheen for excellent technical assistance, and Stefan Schulz for his help in preparing the manuscript. We are grateful to Frans van Leusden (National Institute of Public Health and the Environment, Bilthoven, Netherlands) who provided cultures from recent food-poisoning cases in the Netherlands and to Gilles Vernaud (Institut de Génétique et Microbiologie, Paris, France) for the gift of B. anthracis DNA. Thanks to Fergus Priest (Heriot-Watt University, Edinburgh, UK) for making available an unpublished manuscript on the population structure of the B. cereus group.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Received 8 September 2004; revised 28 September 2004; accepted 14 October 2004.



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