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Applied and Environmental Microbiology, July 2006, p. 5118-5121, Vol. 72, No. 7
0099-2240/06/$08.00+0     doi:10.1128/AEM.00170-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

SHORT REPORT

Characterization of Emetic Bacillus weihenstephanensis, a New Cereulide-Producing Bacterium{dagger}

Line Thorsen,1 Bjarne Munk Hansen,2 Kristian Fog Nielsen,3 Niels Bohse Hendriksen,2 Richard Kerry Phipps,3 and Birgitte Bjørn Budde1*

Department of Food Science, Food Microbiology, Centre for Advanced Food Studies (LMC), The Royal Veterinary and Agricultural University, Frederiksberg, Denmark,1 Department of Environmental Chemistry and Microbiology, National Environmental Research Institute, Roskilde, Denmark,2 The Mycology Group, BioCentrum, Technical University of Denmark, Kgs. Lyngby, Denmark3

Received 23 January 2006/ Accepted 20 April 2006


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ABSTRACT
 
Cereulide production has until now been restricted to the species Bacillus cereus. Here we report on two psychrotolerant Bacillus weihenstephanensis strains, MC67 and MC118, that produce cereulide. The strains are atypical with regard to pheno- and genotypic characteristics normally used for identification of emetic B. cereus strains. MC67 and MC118 produced cereulide at temperatures of as low as 8°C.


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INTRODUCTION
 
Bacillus cereus can cause food-related diarrhea through the production of the nonhemolytic and hemolytic enterotoxin complexes, Nhe and Hbl, respectively, and emesis through the production of the toxin cereulide (D-O-Leu-D-Ala-l-O-Val-L-Val)3 (14). Ehling-Schulz et al. (8) demonstrated that cereulide formation by B. cereus is restricted to a single evolutionary lineage of mesophilic strains, and the genetic determinants are located on a plasmid, pBCE4810 (10). Recently, one emetic psychrotolerant B. cereus strain has been reported (2). However, whether this psychrotolerant strain is a Bacillus weihenstephanensis strain (24) was not specified (2). The increasing demand for convenience foods such as cooked, chilled, ready-to-eat foods raises the question of whether psychrotolerant B. cereus and B. weihenstephanensis present a health risk in these food products because of their ability to survive heat treatment and grow at refrigeration temperatures (7, 30). The objectives of the current work were to investigate the occurrence of cereulide producers among 921 environmental isolates of the B. cereus group, to characterize the cereulide producers with regard to psychrotolerance, and to compare them to well-known cereulide producers at the pheno- and genotypic levels.


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Screening for cereulide producers.
 
A total of 921 B. cereus group isolates (Table 1) were screened by a PCR assay for the emetic character (9), using DNA prepared as described previously (19). Only two strains, MC67 and MC118, showed the emetic character. The two strains originated from different soil samples (within 1 m2) at the same location, a sandy loam on the island of Møn, Denmark (20).


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  		TABLE 1. B. cereus group isolates used in the emetic screeninga


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Identification of emetic Bacillus weihenstephanensis.
 
The identified emetic strains, MC67 and MC118, both grew at 6°C and not at 43°C on brain heart infusion (BHI) agar (Oxoid). PCR analysis (12, 34) revealed that the strains possessed the 16S rRNA gene signature for psychrotolerance and the cold shock protein gene cspA. Thus, MC67 and MC118 should be affiliated with B. weihenstephanensis strains (24), and to our knowledge they are the first strains of this species that have been shown to be emetic.


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Typing and sequencing of emetic Bacillus weihenstephanensis strains.
 
Ehling-Schulz et al. (8) suggested that random amplified polymorphic DNA (RAPD) PCR typing may be useful for rapid identification of potential emetic strains. RAPD_1 PCR (26) and profile analysis with Bionumerics version 1.01 (Applied Maths, Kortrijk, Belgium), using the parameters described elsewhere (8), showed that MC67 and MC118 were identical but were different from the mesophilic emetic strains (Table 2) and from 20 randomly chosen nonemetic B. weihenstephanensis strains (results for 10 strains are shown in Fig. 1). The RAPD_1 profiles were not suitable for rapid identification of psychrotolerant emetic strains. More RAPD profiles of emetic B. weihenstephanensis strains from other origins are required to show whether RAPD typing is a useful screening tool for identification of potential emetic psychrotolerant strains. Sequence analysis of multiple genes of mesophilic emetic B. cereus originating from different countries has shown high similarity between strains (8). Analysis of PCR-amplified DNA sequences (8, 29), using Clustal W (31), of the 16S rRNA gene (1,580 nucleotides [nt]), the 16 to 23S rRNA gene spacer (791 nt), and the spoIIIAC-spoIIIAB sporulation gene fragments (547 nt) as well as the partial cereulide peptide synthetase gene cesB (1091 nt) showed that MC67 and MC118 are 100% identical. The partial cesB gene was amplified as proposed by Ehling-Schulz et al. (11), with the modifications of changing the annealing temperatures to 50°C during the first five cycles and increasing the last 25 cycles to 30 cycles. Purification of DNA and sequencing were as described previously (35). The GenBank accession numbers used for comparison with the 16S rRNA, the 16 to 23S rRNA, and the spoIIIAC-spoIIIAB gene sequences of MC67/MC118 were Z84575 to -94 and Y18473 (24); AJ577274 to -92, AJ578036, AY920248 to -50, and AY920252 to 3 (5); AY758318 to -37 and AY758342 to -49 (8); AY277557 (15); AB021199 (13); AF290547 (32); AE016877 (21); and AM062685 to -6, AE017225, AE017334, and AE017355. The sequence analysis using Clustal W (31) showed that MC67 and MC118 were more related (but not identical) to the psychrotolerant B. weihenstephanensis and B. mycoides strains than to the mesophilic B. cereus group strains (B. thuringiensis, B. anthracis, and B. cereus), including the clonal group of mesophilic emetic strains (8). The 16S rRNA and 16 to 23S rRNA gene sequences of MC67 and MC118 differed by 1 and 1 to 3 nt from the respective sequences of psychrotolerant B. mycoides (the spoIIIAC-spoIIIAB sequences of B. mycoides are not available). The 16S rRNA, the 16 to 23S rRNA, and the spoIIIAC-spoIIIAB gene sequences of MC67/MC118 differed by 1 to 2, 2, and 7 nt from the respective sequences of B. weihenstephanensis; by 5 to 7, 19 to 23, and 49 to 61 nt from those of B. cereus (emetic and nonemetic); by 5 to 6, 21, and 59 nt from those of B. thuringiensis; and by 7 to 8, 21, and 59 nt from those of B. anthracis. Thus, the sequence data suggest that MC67 and MC118 are closely related to B. weihenstephanensis and B. mycoides. However, MC67 and MC118 are most likely B. weihenstephanensis strains, taking into consideration the colony morphology, the fact that other species of the B. cereus group such as B. cereus are heterogeneous (8) and display more or less sequence variability in similar genes between strains, and the limited number of B. weihenstephanensis and B. mycoides sequences available in the databases. The cesB gene, which is a peptide synthetase gene involved in cereulide production, is highly conserved (single nucleotide difference) in mesophilic emetic B. cereus strains, indicating a relatively recent acquisition of the emetic genes (8). Interestingly, the cesB gene fragment of MC67 and MC118 showed only 92% identity to the cesB gene from F4810/72 (GenBank accession number AY691650) (11). The translated CesB amino acid sequence was highly conserved at the N-terminal half, while the C-terminal half was variable (see Table S1 in the supplemental material). The variation in the cesB gene between the psychrotolerant and the mesophilic strains suggest that their separation is not a recent event. The cesB gene is located on a plasmid in mesophilic emetic B. cereus (10), and thus transfer of the emetic plasmid to other bacteria is possible and needs to be further investigated.


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  		TABLE 2. Emetic B. cereus reference strains used


Figure 1
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  		FIG. 1. RAPD_1 profiles of the emetic B. weihenstephanensis strains MC67 and MC118 compared to those of emetic B. cereus strains and nonemetic B. weihenstephanensis strains from a sandy loam on the island Møn in Denmark. Lanes 1 and 2, emetic B. cereus strains F5881 and NS117, respectively; lanes 3 to 5, nonemetic B. weihenstephanensis strains MC73, MC59, and MC8, respectively; lanes 6 and 7, emetic B. weihenstephanensis strains MC67 and MC118, respectively; lanes 8 to 12, nonemetic B. weihenstephanensis strains MC84, MC37, MC10, MC58, and MC17, respectively.


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Examination for amylase activity, salicin fermentation, hemolysis, and enterotoxins.
 
MC67 and MC118 differed from the mesophilic emetic isolates (Table 3) with regard to some of the traditional phenotypic characteristics of emetic strains, as analyzed by methods described elsewhere (27). Further, they differed genotypically by harboring the Hbl enterotoxin complex genes hblA and hblD (8). Our results highlight the precautions which need to be taken when screening for emetic isolates based upon phenotypic traits such as starch hydrolysis and salicin fermentation. Andersson et al. (3) proposed lack of hemolysis as an indicator for emetic strains, and this is also in accordance with our results using the proposed method (3). PCR examination for the enterotoxin genes hblA, hblC, and hblD was performed as described elsewhere (16). The L2 component (HblC) could not be detected using the BCET-RPLA kit as recommended by the manufacturer (Oxoid), using a growth temperature of 32°C.


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  		TABLE 3. Characteristics of the emetic B. weihenstephanensis strains MC67 and MC118 compared to those of emetic B. cereus reference strains


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Cereulide production at different temperatures.
 
Production of cereulide at refrigeration temperatures is critical in relation to food safety, since cereulide will not be destroyed during food processing. To evaluate the risk of cereulide production, MC67, MC118, and the mesophilic strains (Table 2) were grown aerobically on BHI agar (Oxoid) for 10 days at 8, 12, 15, and 25°C. Cereulide was extracted from bacterial mass with 96% ethanol and sonication for 30 min. Cell debris was removed at 17,000 x g for 5 min. Liquid chromatography-high-resolution mass spectrometry (LC-HR-MS) for verification and quantification of cereulide was performed essentially as described elsewhere (17), using the equipment described previously (25) (for details, see Appendix). The MS in-source fragmentation spectrum which was obtained from the ethanol extracts of MC67 and MC118 could be superimposed on the spectrum of the emetic reference strain F4810/72. All the ca. 40 major ions originating from cleavage of the peptide and ester bonds were in the same ratios (results not shown), which indicates that the compound produced by MC67 and MC118 is similar to cereulide. The biological activity of cereulide produced by MC67 and MC118 was confirmed by measurement of the metabolic activity of Chinese hamster ovary (CHO-K1) cells upon exposure to heated ethanol extracts (heated for 10 min at 100°C) as described elsewhere (6), using the WST-1 cell proliferation assay as described by the manufacturer (Roche, Hvidovre, Denmark). MC67 and MC118 were the only emetic strains that were able to grow and produce cereulide at 8°C (Table 4). Compared to the mesophilic strains, MC67 and MC118 produced large amounts of cereulide at 25°C, which indicates no coherence between temperature growth profile and cereulide production (Table 4). Cereulide was not produced at critical concentrations for food poisoning (23) at temperatures of 8 to 15°C. However, unknown factors, such as temperature abuse, the food matrix, and interactions with other bacteria, which were not tested in this work might provoke cereulide production. Therefore, the risk of food poisoning from psychrotolerant emetic strains in refrigerated foods needs to be further investigated.


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  		TABLE 4. Cereulide production by B. weihenstephanensis MC67 and MC118 compared to that of reference strains of emetic B. cereusa


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Nucleotide sequence accession numbers.
 
The identical sequences of MC67 and MC118 were deposited in GenBank under accession numbers DQ345789, DQ345790, DQ345791, and DQ345792 for spoIIIAC-spoIIIAB, cesB, the 16S rRNA gene, and the 16 to 23S rRNA gene, respectively.


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APPENDIX
 
LC-HR-MS was performed using an Agilent Zorbax SB-CN column (150 by 2 mm [inner diameter] by 5 µm) and the equipment described previously (25). A linear water-CH3CN gradient system (H2O buffered with 10 mM HCOONH4 and 20 mM HCOOH and CH3CN buffered with 20 mM HCOOH) at a flow rate of 0.3 ml/min was used, starting at 50% CH3CN, increasing to 100% for 12 min, and staying at 100% for 3 min before reverting to the starting conditions. Samples were analyzed in electrospray ionization positive mode at a resolution of >7,000 (half peak height) (25) and with data being centroid spectra from m/z 200 to 1,500. Three scan functions (1 s each) were used: (i) with a potential difference between the skimmers of 50 to 60 V (no fragmentation), (ii) with a difference of 100 to 125 V (high fragmentation), and (iii) the spray from the lock spray probe (second electrospray ionization spray) for on-line mass correction. The responses of valinomycin and cereulide in LC-HR-MS have been shown to be very similar (17). Valinomycin was used as an internal standard at 0.82 µg/ml. Cereulide and valinomycin were detected from the first scan function of their reconstructed ion chromatograms (±m/z 0.05) of the ammoniated adducts (M + NH4)+ at m/z 1,170.7125 and 1,128.6655, respectively. The detection limit (on column, first scan function) for valinomycin was ca. 80 pg/2 µl at a signal-to-noise ration of 10. The identity of cereulide in the samples was confirmed from the second scan function which gave significant in-source fragmentation (>40 ions) to validate the primary structure of the depsipeptide.


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ACKNOWLEDGMENTS
 
This work has been financially supported by the Danish Bacon and Meat Council, Copenhagen, Denmark.

The collaboration with the Danish Meat Research Institute, Roskilde, Denmark, is highly appreciated.


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FOOTNOTES
 
* Corresponding author. Mailing address: The Royal Veterinary and Agricultural University, Department of Food Science, Rolighedsvej 30, 4th Floor, DK-1958 Frederiksberg C, Denmark. Fax: 45 35 28 32 14. Phone: 45 35 28 32 84. E-mail: birgitte.budde{at}privat.dk. Back

{dagger} Supplemental material for this article may be found at http://aem.asm.org/. Back


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Applied and Environmental Microbiology, July 2006, p. 5118-5121, Vol. 72, No. 7
0099-2240/06/$08.00+0     doi:10.1128/AEM.00170-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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