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Journal of Clinical Microbiology, August 2002, p. 3053-3056, Vol. 40, No. 8
0095-1137/02/$04.00+0     DOI: 10.1128/JCM.40.8.3053-3056.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Enterotoxigenic Profiles of Food-Poisoning and Food-Borne Bacillus cereus Strains

Marie-Hélène Guinebretière,* Véronique Broussolle, and Christophe Nguyen-The

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

Received 6 December 2001/ Returned for modification 25 February 2002/ Accepted 3 May 2002


   ABSTRACT
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The enterotoxigenic profiles of 51 B. cereus food-related strains were compared to those of 37 B. cereus food-poisoning strains. cytK and association of hbl-nhe-cytK enterotoxin genes were more frequent among diarrheal strains (73 and 63%) than among food-borne strains (37 and 33%). Unlike diarrheal strains, food-borne strains showed frequent nhe and hbl gene polymorphisms and were often low toxin producers.


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Bacillus cereus can cause food-borne diarrhea by producing heat-labile enterotoxins during growth of vegetative cells in the small intestine (6). Four different enterotoxins have been characterized: two protein complexes, hemolysin BL (HBL) and nonhemolytic enterotoxin (NHE), and two enterotoxic proteins, enterotoxin T (bc-D-ENT) (1) and cytotoxin K (11). HBL complex is composed of three proteins, B, L1, and L2 (4, 5) transcribed from the genes hblC (encoding L2), hblD (encoding L1), and hblA (encoding B), organized in one operon together with a fourth gene, hblB (encoding the B' protein) (9, 13). NHE complex is also composed of three different proteins, NheA, NheB, and NheC encoded by the three genes nheA, nheB, and nheC, and it is also organized in one operon (7).

B. cereus is widespread in foods, whereas diarrheal poisoning caused by B. cereus is fairly infrequent. The ability to cause diarrhea thus presumably varies among strains. Only few data are available concerning the genetic and toxigenic potential of B. cereus strains from vegetable products, although vegetables are frequently contaminated by B. cereus. The objective of the present work was to analyze the distribution of enterotoxin genes and enterotoxin production in food strains of various origins (isolated from cooked chilled foods and vegetables) and to compare enterotoxigenic profiles to those in diarrheal food-poisoning strains.

Eighty-eight bacterial strains (51 food-related strains and 37 food-poisoning strains, listed below in Tables 2 and 3) were tested for enterotoxin production, using the B. cereus Enterotoxin Reverse Passive Latex Agglutination test kit (Oxoid, Basingstoke, England) and the Bacillus Diarrheal Enterotoxin visual immunoassay (Tecra Diagnostics, Roseville, Australia), according to the manufacturers' instructions. Amounts of produced enterotoxin were evaluated with index values derived from the Oxoid and Tecra reading scale (Table 2). All strains were also tested for the presence of the genes hbl (C, D, A, and B), nhe (A, B, and C), bceT (encoding the bc-D-ENT enterotoxin), and cytK (encoding cytotoxin K). DNA was extracted as previously described (2). The primers used for the detection of the various genes are presented in Table 1. Amplified products from strains ATCC 14579T, 1230/88, and 391/98 were sequenced to check the specificity of the designed primers. Except for amplifications with hblB primers, the PCR mixture (50 µl) consisted of 100 ng of DNA template, 200 µM deoxynucleoside triphosphate mix (Eurogentec S. A., Seraing, Belgium), 2.5 mM MgCl2, a 500 nM concentration of each primer, 0.75 U of AmpliTaq polymerase (Perkin-Elmer, Courtaboeuf, France), and AmpliTaq buffer (Eurogentec). Thermal cycling was carried out in a PCR 9700 thermocycler (Perkin-Elmer) with the following run: a starting cycle of 2 min at 94°C, followed by 35 cycles of 1 min at 94°C, 1 min at the annealing temperature (Table 1), and 2 min at 72°C, and a final extension of 5 min at 72°C. For the hblB amplification reaction (50 µl), 500 µM deoxynucleoside triphosphate mix, 5 mM MgCl2, and a 300 nM concentration of each primer were used instead of the concentrations stated above. Parameters determined with the PCR 9700 thermocycler were 2 min at 94°C followed by 10 cycles of 10 s at 94°C, 30 s at 58°C, and 2 min at 68°C; 20 cycles of 10 s at 94°C, 30 s at 58°C, 2 min (plus 20 s per cycle) at 68°C; and a final extension at 68°C for 7 min. Strains that were negative in PCR experiments were submitted to Southern blotting to check for the absence of hbl, nhe, and cytK. Positive controls were included for each tested probe. Five micrograms of genomic DNA was digested to completion with EcoRI and electrophoresed overnight on a 1% agarose gel at 15 V. Southern blotting was carried out according to standard protocols (14). Probes were generated by PCR amplifications of hblC, hblD, hblA, nheA, nheB, nheC, and cytK gene fragments, and 32P-labeling was performed with Ready-to-Go DNA labeling beads (Amersham Pharmacia Biotech, Orsay, France) according to the manufacturer's recommendations. Membranes were hybridized at 60°C for 2 h in Rapid hybridization buffer (Amersham Pharmacia Biotech), washed according to supplied protocols, and exposed to phosphorimager screens (Storm; Molecular Dynamics, Bondoufle, France) before revelation. The Kolmogorov-Smirnov two-sample test was used to determine whether the frequency and association of genes and the production of enterotoxins were significantly different between food-poisoning strains and food-borne strain groups. The test was performed using SYSTAT (version 9.0; SPSS Inc., Chicago, Ill.).


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  		TABLE 2. Characteristics of food-poisoning strains

 

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  		TABLE 3. Characteristics of food-borne strains

 

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  		TABLE 1. Characteristics of primers used in this studya

 
Detection of enterotoxin genes. For the 88 B. cereus strains, hbl and nhe gene detection by both PCR and Southern blotting methods correlated well with the results of the Oxoid and Tecra tests, respectively (Tables 2 and 3). Among the 37 food-poisoning strains, none needed confirmation by Southern analysis for hbl genes (C, D, and A), which were easily detected by PCR, and only eight strains (22%) negative in PCR for one or two of the nhe genes were found to be positive by Southern blotting (Table 2). This contrasted with results obtained for the 47 and 49 food-borne strains that were positive for the HBL and NHE enterotoxins, respectively: 17 strains (36%) and 31 strains (63%) negative in PCR for one or two of the respective hbl and nhe genes were found to be positive by Southern blotting (Table 3). This indicates a high polymorphism in the hbl and nhe sequences among food-associated strains, which differed markedly from food-poisoning strains in that respect. Southern analysis showed that genetic heterogeneity among B. cereus strains, and particularly among environmental strains, is more truly associated with sequence polymorphism than with the lack of one of the genes composing the hbl or nhe operon.

Distribution of enterotoxin genes. Almost all the strains from the food-poisoning and food-borne ecosystems carried nhe genes, as observed in previous works (8, 12). The bceT gene was widely distributed (57 and 71% among food-poisoning and food-borne strains, respectively). The hbl (C, D, and A) genes were also highly frequent in the two groups of strains (73 to 92%). The cytK gene was frequently detected among diarrheal strains, 73% of which carried it. It was present at a significantly (P = 0.007) lower frequency among food-borne strains; only 37% of the food strains were concerned. Stenfors and Granum (15) found three strains (13%) carrying this gene among 23 strains from dairy products. Profiles associating the nhe, hbl, and cytK genes were significantly (P = 0.049) more frequent among food-poisoning strains (63% of the strains concerned) than among food-borne strains (33% of the strains).

Enterotoxin levels produced among strain samples. Among strains carrying hbl genes, the percentage of those producing high levels of enterotoxin HBL (with an index of >=128) was significantly (P = 0.002) higher for food-poisoning strains (74%) than for food-borne strains (32%). Similarly, among strains carrying nhe genes, the frequency of high enterotoxin NHE producers (production at an index of >=4) was 78% for food-poisoning strains and only 20% for food-related strains. Furthermore, 11 strains (22%) among the 49 food-borne strains carrying nhe genes did not produce or only weakly produced enterotoxin NHE (index < 3). This emphasizes a difference in gene expression between food-borne and food-poisoning strains. Psychrotrophic strains isolated from pasteurized milk by in't Veld et al. (10) were also low producers of enterotoxin BL, with only 26 strains out of 86 (30%) exhibiting an index of >=128 in the Oxoid test. Beattie and Williams (3) also observed wide variations in the amount of enterotoxin produced from dairy-associated strains.

Some significant differences were found in the genetic and toxigenic potential of food-poisoning strains and food-borne strains, supporting the hypothesis that many food-borne strains are less prone to cause diarrhea.

StrainhblChblDhblAhblBkit testnheAnheBnheCtest indexcytK,bceTSZ++++4+s++s3-+ SB'++++>128+s++4-+ SD+++-16+s++3.5-+ SM++++64+s++s2.5-+ C41+s++-2+s++1.5-+ I6+s+s+s-2+s++s4-+ I11+++-128+++3.5-+ I20+s++-16++s+3-+ I21+s+s+-16+s++3-+ C43+++-128+++s5+- I22+++-128+++4+- 32++++>128+s++4++ BC'++++64++s+3++ A3++++16+s++3.5++ C3++++128+++3.5++ C15++++128+++3.5++ C38+++->128+++4++ C57+++-32+++3.5++ I2+++-64+++3.5++ I7+s++-64+++3.5++ I10+++-128+++3.5++ I12+++->128+s++3.5++ I13+s++-128+++5++ I16+++->128++s+3.5++ I17+++-128+++2.5++ I23++++32+s++2.5++


   ACKNOWLEDGMENTS
 
This work was supported by specific funds from the Département Transformation des Produits Végétaux (INRA, France) and by a research project, AQS R0013 (Ministère de l'Agriculture et de la Pêche, Ministère de la Recherche, France).

We thank P. E. Granum (The Norwegian School of Veterinary Science, Oslo, Norway), M. A. Andersson and M. S. Salkinoja-Salonen (University of Helsinki, Helsinki, Finland), J. Mc Lauchlin (PHLS, London, England), C. Denis (ADRIA Normandie, Villers Bocage, France), and M. L. De Buyser (AFSSA, Maisons-Alfort, France) for providing strains. We also thank D. Lereclus (Institut Pasteur, Paris, France) for providing the sequence of the cytK gene obtained from Bacillus thuringiensis. We are very grateful to S. Courtillier for help in the realization of immunoassays.


   FOOTNOTES
 
* Corresponding author. Mailing address: INRA, UMR A408 INRA-Université d'Avignon, Domaine St-Paul, Site Agroparc, 84914 Avignon Cedex 9, France. Phone: (33) 4 32 72 25 24. Fax: (33) 4 32 72 24 92. E-mail: guinebre{at}avignon.inra.fr. Back


   REFERENCES
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Journal of Clinical Microbiology, August 2002, p. 3053-3056, Vol. 40, No. 8
0095-1137/02/$04.00+0     DOI: 10.1128/JCM.40.8.3053-3056.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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