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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References

Bacillus cereus is one of the important food pathogens. Since B. cereus group cells, such as B. cereus, B. thuringiensis, B. anthracis and B. mycoides, share many phenotypical properties and a high level of chromosomal sequence similarity, it is interesting to investigate the virulence profiles for B. cereus group cells, including B. cereus strains isolated from foods and samples associated with food-poisoning outbreaks. For this investigation, the presence of enterotoxin genes, such as those of haemolysin BL, B. cereus enterotoxin T and enterotoxin FM, were assayed by polymerase chain reaction (PCR) methods. Meanwhile, their enterotoxin activities were assayed using the BCET-RPLA kit, haemolytic patterns on sheep blood agar and their cytotoxicity to Chinese hamster ovary (CHO) cells. Results showed that there were 12 enterotoxigenic profiles for the 98 B. cereus group strains collected. In addition, if any of the three types of enterotoxins was present in the B. cereus group cells, these cells were shown to be cytotoxic to the CHO cells. Similar enterotoxigenic profiles could be found among strains of B. cereus, B. mycoides and B. thuringiensis. Thus, all B. cereus group strains may be potentially toxigenic and the detection of these cells in foods is important. We thus designed PCR primers, termed Ph1/Ph2, from the sphingomyelinase gene of B. cereus cells. These primers were specific for all B. cereus group strains and could be used for the detection of B. cereus cells contaminated in food samples.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References

Bacillus cereus is one of the important food pathogens. It may cause two distinct poisoning syndromes, i.e. diarrhoea and emesis, and be responsible for some non-gastrointestinal infections ( Kramer & Gilbert 1989; Drobniewski 1993). This organism is often isolated from food samples, such as raw milk, dairy products and rice products, as a contaminant ( Kramer & Gilbert 1989; Larsen & Jørgensen 1997). It is a spore-forming bacterium and is widespread in the environment. Granum (1994) has reported that there are several different toxins produced by B. cereus: enterotoxins, haemolysin (cereolysin, haemolysin II and sphingomyelinase), phospholipase C (phosphophatidylinositol hydrolase, phosphatidylcholine hydrolase and sphingomyelinase) and emetic toxin. Three different types of B. cereus enterotoxins have been purified and their genes cloned, haemolysin BL (HBL) ( Heinrichs et al. 1993 ; Beecher et al. 1995 ; Ryan et al. 1997 ), B. cereus enterotoxin T (BceT) ( Agata et al. 1995 ) and enterotoxin FM (EntFM) ( Asano et al. 1997 ).

Since it has been reported that four species of B. cereus group cells, i.e. B. cereus, B. thuringiensis, B. mycoides and B. anthracis, share many phenotypical properties, some workers have questioned the taxonomic inter-relationship of these species and also the status of these species as separate species ( Carlson & Kolstø 1993; Carlson et al. 1994 ; Helgason et al. 1998 ). DNA-DNA hybridization studies on strains of B. anthracis, B. cereus and B. thuringiensis also show that these organisms share relatively high levels of chromosomal base sequence similarity ( Somerville & Jones 1972; Drobniewski 1993). In addition, the primary sequences of the 16S rRNA genes of these Bacillus species exhibit very high levels (> 99%) of sequence similarity ( Ash et al. 1991a,b ). Based on these facts, we were thus interested in the characterization of the enterotoxigenic profiles for B. cereus group cells, including 58 B. cereus strains isolated from food samples and samples associated with food-poisoning outbreaks.

To characterize the enterotoxigenic properties for the B. cereus group strains collected in our laboratory, the polymerase chain reaction (PCR) primers reported by Mäntynen & Lindström (1998) for the hblA gene, the ETF/ETR primers developed by Agata et al. (1995) for the bceT gene and the ENTA/ENTB primers reported by Asano et al. (1997) for the entFM gene were used. Furthermore, the presence of HBL and other enterotoxins in these B. cereus group strains was assayed by the haemolytic patterns on the blood agar plate ( Beecher & Wong 1994a), the BCET-RPLA toxin kit and the cytotoxicity to Chinese hamster ovary (CHO) cells. Results show that B. cereus group cells are potentially toxigenic and thus a method to detect all the B. cereus group cells is important.

The Conventional Bacteriological Analytical Manual method for the detection of B. cereus cells is dependent on the use of selective media as well as biochemical tests and may take 2–3 d ( FDA 1995). Furthermore, it is difficult to detect low numbers of B. cereus cells in foods ( Schraft & Griffiths 1995). Therefore, we developed a set of PCR primers based on the sphingomyelinase gene (sph). These PCR primers are specific to all B. cereus group strains tested. Application of this primer set for the detection of B. cereus cells contaminated in food samples was successful.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References

Bacterial strains

Twenty-six strains of B. cereus, two strains of B. anthracis, three strains of B. mycoides, nine strains of B. thuriengiensis, 18 strains of other Bacillus spp. and several non-Bacillus spp. strains collected from different sources were used in this study. In addition, 28 B. cereus strains (strain BCN 1–28) from food samples and 30 other B. cereus strains (BCN 29–58) from samples associated with food-poisoning outbreaks, such as human body, water, kitchen-ditch, dishcloth and chopping-board, were also used ( Table 1).

Table 1.  Bacterial strains used in this study
SpeciesStrain no. and sources
  1. ATCC, American Type Culture Collection, Rockville, MD, USA; CCRC, Culture Collection and Research Center, Taiwan; CDC, Center for Disease Control and Prevention, Atlanta, GA, USA; USDA, United States Department of Agriculture, Washington, DC, USA; NLFD, National Laboratory of Foods and Drugs, Taipei, Taiwan; IPM, Institute of Preventive Medicine, Taichung-Branch, Taiwan; TACTRI, Taiwan Agricultural Chemical and Toxic Substances Research Institute, Taichung, Taiwan.

Bacillus cereus group
B. cereusBC1 (CCRC 10250), BC2 (CCRC 10282), BC3 (ATCC 14579), BC4 (ATCC 13061), BC5 (CCRC 11026), BC6 (CCRC 11835), BC7 (ATCC 7004), BC8 (CCRC 11833), BC9 (CCRC 11834), BC10 (CCRC 11910), BC11 (CCRC 11913), BC12 (ATCC 11778), BC13 (ATCC 21182), BC14 (ATCC 21281), BC15 (ATCC 21282), BC16 (ATCC 21769), BC17 (ATCC 21771), BC18 (ATCC 19637), BC19 (CCRC 14194), BC20 (CCRC 14195), BC21 (CCRC 14196), BC22 (CCRC 14197), BC23 (CCRC 14655), BC24 (CCRC 14689), BC25 (CCRC 14699), BC26 (ATCC 49064), laboratory isolates from NLFD, BCN 1–28, food-poisoning-associated outbreak isolates from IPM, BCN 29–58
B. thuringiensisBT1 (CCRC 14380), BT2 (CCRC 14381), BT3 (ATCC 10729), BT4 (CCRC 11502), BT5 (CCRC 15070), BT6 (TACTRI 1), BT7 (TACTRI 2), BT8 (CCRC 14377), BT9 (CCRC 14616)
B. mycoidesBMY1 (ATCC 6462), BMY2 (ATCC 10206), BMY3 (CCRC 11968)
B. anthracisBA1 (ATCC 8705), BA2 (ATCC 14578)
Other Bacillus spp.B. subtilis (CCRC 10029, CCRC 10258, CCRC 10267), B. stearothermophilus (CCRC 10285, CCRC 11029, CCRC 10610), B. brevis (ATCC 8186), B. coagulans (CCRC 10251), B. megaterium (ATCC 14581), B. lentus (ATCC 10840), B. licheniformis (ATCC 14580), B. circulans (ATCC 4513), B. polymyxa (ATCC 842), B. amylolique (ATCC 23350), B. pumilus (ATCC 7061), B. firmus (ATCC 14575), B. psychrophilus (ATCC 23304), B. sphaericus (ATCC 14577)
Non-Bacillus spp.Staphylococcus aureus (CCRC 12654, 12656, 12657, 12600), Staph. epidermidis (CCRC 11030), Escherichia coli (ATCC 25922), Morganella morganii (CCRC 10706), Proteus vulgaris (CCRC 10728), Yersinia enterocolitica (CCRC 10807), Enterobacter aerogenes (CCRC 10370), Erwinia carotovora (CCRC 11298), Klebsiella pneumoniae (CCRC 10629), Serratia marcescens (CCRC 10948), Citrobacter mamlonaticus (ATCC 25405), Lactobacillus brevis (CCRC 12187), Lact. delbrueckii subsp. lactis (CCRC 12256), Lact. buchneri (CCRC 14066), Listeria monocytogenes (USDA ScottA), L. ivanovii (CDC 1714), Streptococcus faecalis (CCRC 10066), Clostridium perfringens (CCRC 10914)

Bacillus cereus group cells were cultivated in Brain Heart Infusion broth (DIFCO, Detroit, MI, USA) plus 0·1% glucose (BHIG broth) overnight at 37 °C with rotary shaking (180 rev min−1). Stock cultures were kept at −70 °C with 20% glycerol.

Polymerase chain reaction primers

The target genes to be detected and the PCR primers used were sph gene, primers Ph1/Ph2; hblA gene, primers HblA1/HblA2; bceT gene, primers ETF/ETR and entFM gene, primers ENTA/ENTB. Except for primers Ph1/Ph2, all the primers have been reported previously ( Agata et al. 1995 ; Mäntynen & Lindström 1998; Asano et al. 1997 ). The sequences, locations within genes, annealing temperatures and sizes of their PCR products for these primers are shown in Table 2.

Table 2.  Polymerase chain reaction (PCR) primers used in this study
Target gene PrimerOligonucleotide sequence (5′−3′) *Location within gene An TM (°C) Product size (bp) Reference
  • *

    F, Forward sequence; R, reverse and complementary sequence.

  • According to the sequences from L20441 for hblA gene, M20194 for sph gene and D17312 for bceT gene.

  • Annealing temperature for PCR.

hblAHblA1/HblA25′-GCTAATGTAGTTTCACCTGTAGCAAC-3′/(F)121–14658874Mäntynen & Lindström (1998)
5′-AATCATGCCACTGCGTGGACATATAA-3′ (R)994–969   
sphPh1/Ph25′-CGTGCCGATTTAATTGGGGC-3′/(F)1007–102658558This study
5′-CAATGTTTTAAACATGGATGCG-3′ (R)1564–1543   
bceTETF/ETR5′-TTACATTACCAGGACGTGCTT-3′/(F)1354–137456428Agata et al. (1995)
5′-TGTTTGTGATTGTAATTCAGG-3′ (R)1781–1761   
entFMENTA/ENTB5′-ATGAAAAAAGTAATTTGCAGG-3′/(F)1–21521269Asano et al. (1997 )
5′-TTAGTATGCTTTTGTGTAACC-3′ (R)1249–1269   

Primers Ph1 and Ph2 were designed by use of the Wisconsin Sequence Analysis Package which was developed by the Genetic Computer Group Inc. (Madison, WI, USA). The DNA sequence of the phospholipase and sphingomyelinase gene of B. cereus (accession no. M20194) was compared with those of other Bacillus spp. and non-Bacillus spp. available in GeneBank/EMBL through the program of FASTA. By such comparison, oligonucleotide primers unique to B. cereus group strains were designed.

Polymerase chain reaction amplification

Polymerase chain reaction mixture (50 μl) contained 200 μmol l−1 each of dATP, dGTP, dTTP and dCTP, 1 × PCR buffer (10 mmol l−1 Tris.HCl, pH 8·8; 1·5 mmol l−1 MgCl2; 50 mmol l−1 KCl; 0·1% Triton X 100), 25 pmol primer set, approximately 50 ng bacterial DNA and 0·4 unit Dynazyme (Finnzyme, Riihitontuntie, Finland). Amplification was performed in a PCR thermal cycler (GeneAmp PCR System 9600; Perkin Elmer, Norwalk, CT, USA). Polymerase chain reaction conditions for primers HblA1/HblA2 were 35 cycles at 94 °C for 20 s, 58 °C for 40 s and 72 °C for 40 s. On the other hand, the bceT, entFM and sph genes were amplified for 35 cycles at 94 °C for 20 s, different annealing temperatures ( Table 2) for 20 s and 72 °C for 20 s. The PCR products were then analysed by 2% agarose gel electrophoresis.

Detection of enterotoxins by BCET-RPLA

BHIG broth (5 ml) was inoculated with one loop of 24 h culture and incubated for 18–20 h at 37 °C. The cultures were centrifuged for 5 min at 5000 rev min−1 (Himac SCR20B; Hitachi, Tokyo, Japan) and the cell-free supernatant fluid subjected to enterotoxin detection. Enterotoxins were detected with the B. cereus enterotoxin (diarrhoeal type) reversed passive latex agglutination test kit (BCET-RPLA) (Denka Seiken, Tokyo, Japan). This kit is specific for the L2 component of HBL enterotoxin ( Beecher & Wong 1994b). The assay was performed according to the manufacturer’s instructions.

Detection of haemolysin BL activity

The methods of Beecher & Wong (1994a) were used. Solid media for the detection of haemolysin BL were prepared as follows. Nutrient agar (DIFCO) was supplemented with 0·15 mol l−1 NaCl, autoclaved and cooled to 50–55 °C. Goat serum (2%) and defibrinated sheep blood (2·5%) were then added and the mixture used for agar plate preparation. All solid media were allowed to set at room temperature before use.

For HBL assay, one loop of the test strain was inoculated into 5 ml BHIG broth and incubated at 37 °C with shaking (180 rev min−1) for 12 h. The supernatant fluid of the bacterial culture was then spotted by touching the end of a micropipet (250 μl capacity) tip on the blood agar plate and the plate was kept at 22 °C for 24–48 h. The haemolytic pattern was then observed. Discontinuous haemolytic zones did not begin at the edge of the colony but some distance away from the edge. A discontinuous haemolytic pattern indicates the presence of HBL in B. cereus group strains.

Chinese hamster ovary cell assay

Cytotoxicity was determined by the detachment of the CHO cells from an established monolayer as described by Gentry & Dalrymple (1980). The CHO cells were maintained in McCoy’s 5A medium supplemented with 10% foetal bovine serum, 1·22% sodium bicarbonate, 100 units ml−1 penicillin and 100 μg ml−1 streptomycin. Freshly trypsinized cells were counted, suspended to the desired concentration in growth medium and 0·1 ml (106 cells ml−1) of the cell suspension pipetted into each of the 96 wells of the microtitre plate. Monolayers were established by 18–20 h incubation at 37 °C under 5% CO2 atmosphere. One loop of the B. cereus cells was also cultured in 5 ml BHIG broth (37 °C, 12 h) and 0·5 ml of such bacterial culture was transferred to 50 ml BHIG broth. After cultivation at 37 °C for 5 h, 0·1 ml of the serial diluted bacterial supernatant fluids was added into the well containing the monolayer of CHO cells. The plates were incubated for 18–24 h at 22 °C. After incubation, detached cells, medium and bacterial supernatant fluid were removed. The remaining cells were fixed with 2% formalin. After 1 min, the fixative was removed and each well stained with 0·13% crystal violet for 20 min. Each well was then rinsed three times with sterile deionized water followed by air-drying. Afterwards, 0·1 ml 50% ethanol was added and the mixture left for 1–2 h. The absorbance was determined at 630 nm using an ELISA reader (EL340; BIO-TEK, Winooski, VT, USA).

Detection of Bacillus cereus group cells in food samples

Polymerase chain reaction primers (Ph1/Ph2) specific for the sph genes of B. cereus group cells were used for the detection of these organisms in food samples, such as whole milk, cooked rice, eggs and pork purchased from local food markets. Minced food samples (50 g) were mixed with 450 ml BHIG broth and homogenized with a stomacher (Stomacher 400; Seward, London, UK) at high speed for 2 min. Sterile water (100 μl) with or without B. cereus group cells was then added to 10 ml of such food homogenate. The inoculation levels of B. cereus cells were equivalent to 100−105 cfu g−1 of the food sample. After 8 h pre-enrichment at 37 °C, 100 μl culture was diluted 10-fold with sterile water and 10 μl of the dilution mixed with 30 μl 4/3 × PCR buffer, 200 μmol l−1 each of dNTP and 25 pmol each of the primers Ph1/Ph2. After heating at 97 °C for 30 min, 10 μl of the polymerase solution (1 × PCR buffer, 0·4 unit Dynazyme) was added and the mixture subjected to PCR according to the conditions described earlier.

Results and discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References

Enterotoxin profiles for Bacillus cereus group cells

As described earlier, B. cereus group cells, especially B. cereus and B. thuringiensis, share many phenotypical properties and relatively high levels of chromosomal base sequence similarity ( Ash et al. 1991a,b ), thus it is interesting to know the enterotoxin profiles for these organisms. Table 3 shows the results for the PCR detection of different enterotoxin genes, the results of immunoassay using enterotoxin specific kits, the patterns of haemolysis on the blood agar plates and the cytotoxicity to the CHO cells for the B. cereus group cells including 58 B. cereus strains isolated from foods and samples associated with food-borne outbreaks.

Table 3.  Detection of the presence of enterotoxins and their genes as well as cytotoxicity for Bacillus cereus group cells
Profile and strainsTotal strains Pattern of haemolysis HblA1/ HblA2 BCET- RPLA ETF/ ETR ENTA/ ENTB CHO cell cytotoxicity §Ph1/Ph2
  • *

    h, Human source; w, water; k, kitchen-ditch; d, dishcloth; c, chopping-board.

  • D, Discontinuous haemolytic pattern; C, continuous haemolytic pattern; −, no haemolytic pattern.

  • + and −, positive and negative polymerase chain reaction results.

  • §

    Reactions were considered positive when ≥50% of the cells had detached.

  • CHO, Chinese hamster ovary.

Bacillus cereus
Profile I13D++++++
 CCRC strains, BC1, BC3, BC5, BC9,  BC10, BC14 and BC24 7       
 Outbreak-associated source,  BCN29(h) *, BCN30(h), BCN34(h),  BCN35(k), BCN41(h) and BCN42(h) 6       
Profile II6D+++++
 CCRC strains, BC8, BC19, BC20,  BC25 and BC26 5       
 Food isolates, BCN121       
Profile III3D+++++
 Food isolates, BCN27 and BCN282       
 Outbreak-associated source, BCN53(c)1       
Profile IV2C++++++
 CCRC strain, BC111       
 Food isolates, BCN11       
Profile V1C+++++
 CCRC strain, BC21       
Profile VI1C++++
 CCRC strain, BC131       
Profile VII2C+++
 CCRC strain, BC121       
 Food isolate, BCN211       
Profile VIII37C+++
 CCRC strains, BC4, BC6, BC7, BC16,  BC17, BC21, BC22 and BC23 8       
 Food isolates, BCN2, BCN5, BCN6,  BCN7, BCN9, BCN10, BCN14,  BCN15, BCN20, BCN22, BCN23,  BCN24 and BCN25 13       
 Outbreak-associated sources, BCN31(h),  BCN33(h), BCN38(c), BCN43(h),  BCN45(h), BCN46(h), BCN47(h),  BCN48(h), BCN49(h), BCN50(h),  BCN51(h), BCN52(w), BCN54(k),  BCN55(d), BCN56(c) and BCN57(h) 16       
Profile IX18C++++
 CCRC strain, BC181       
 Food isolates, BCN3, BCN4, BCN8,  BCN11, BCN13, BCN16, BCN17,  BCN18, BCN19 and BCN26 10       
 Outbreak-associated sources,  BCN32(h), BCN36(h), BCN37(k),  BCN39(h), BCN40(h), BCN44(h) and  BCN58(h) 7       
Profile X1C+
 CCRC strain, BC15
 B. cereus group cells except B. cereus
Profile II1D+++++
 BMY22
Profile III1D+++++
 BMY1
Profile I7D++++++
 BT3, BT4, BT5, BT6, BT7, BT8 and  BT9
Profile XI1C+++++
 BT2
Profile VII1C+++
 BMY3
Profile VIII1C+++
 BT1
Profile XII2+
 BA1 and BA2

Primers HblA1/HblA2 were designed for the specific detection of the hblA gene encoding the B component of HBL of B. cereus group cells ( Mäntynen & Lindström 1998). Only 26 of the 84 B. cereus cells collected in our laboratory generated the expected PCR product with molecular weight equal to 876 bp. Of the 28 B. cereus strains isolated from food samples, four strains were hblA PCR-positive while, of the 30 B. cereus strains isolated from samples associated with outbreaks, seven strains were hblA PCR-positive. Thus, no significant difference was found for strains from these two sources. Seven B. thuringiensis strains and two B. mycoides strains also generated the expected PCR products. However, two B. anthracis strains tested were found to be PCR-negative.

Beecher & Wong (1994a) reported that the discontinuous haemolytic pattern produced on sheep and calf blood agar was a distinct characteristic for HBL. In this study, the haemolytic patterns for all B. cereus group strains collected in our laboratory were analysed. Twenty-three of the 26 hblA PCR-positive B. cereus strains showed the discontinuous haemolytic patterns. The remaining three strains showed continuous patterns. All the hblA PCR-negative B. cereus strains showed continuous haemolytic patterns. However, seven of the eight hblA PCR-positive B. thuringiensis strains and two hblA PCR-positive B. mycoides strains also showed the discontinuous haemolytic patterns. In contrast, a haemolytic zone on sheep blood agar was not found for B. anthracis strains ( Table 3). Thus, the presence of the hblA gene seems to be highly correlated to the discontinuous haemolytic patterns as reported by Beecher & Wong (1994a).

When the B. cereus and B. cereus group strains shown in Table 3 were assayed with the BCET-RPLA enterotoxin kit (Denka Seiken), it was found that all the 36 hblA PCR-positive strains, including 26 B. cereus strains, eight B. thuringiensis strains and two B. mycoides strains, were also enterotoxin-producing strains, except for strain BC13 which was hblA PCR-positive but BCET-RPLA-negative. All the hblA PCR-negative strains were negative as assayed with this enterotoxin kit. The above observations indicate that the results from the BCET-RPLA assay agree with the results of the hblA PCR assay except for strain BC13. In addition, several B. cereus and other B. cereus group strains, such as strains BC2, BC11 and BT2, although they were hblA PCR- and BCET-RPLA-positive, showed the continuous haemolytic pattern on the blood agar plate. Although it has been reported that the discontinuous haemolytic pattern was only transient for B. cereus cells ( Beecher & Wong 1994a), discontinuous patterns were not found for strains BC2, BC11 and BT2 throughout the whole assay process.

Beecher & Wong (1994b) reported that the BCET-RPLA kit detects the L2 component of HBL, but Mäntynen & Lindström (1998) reported that the primers HblA1/HblA2 used here detected the B component of the hblA gene. Bacillus cereus strains deficient in part of the HBL components have also been reported ( Mäntynen & Lindström 1998). Strain BC13 might be L2 component-deficient and thus was PCR-positive but BCET-RPLA negative. It may also be possible that the expression level of the hblA gene from strain BC13 was below the detection limit of the BCET-RPLA kit. For strains BC2 and BC11, although both the PCR and RPLA assays showed positive results, the concentrations of each HBL component produced might not be enough or the ratio of each component might not be correct to generate the discontinuous haemolytic patterns. Formation of the discontinuous pattern might need a minimum concentration and defined ratios for each component of the HBL enterotoxin ( Beecher & Wong 1994b). The HBL is a tripartite enterotoxin that requires all three components for maximum activity ( Beecher et al. 1995 ). Furthermore, polymorphism of the haemolysin B-component gene within the amplified PCR products has been found for the B. cereus group cells ( Mäntynen & Lindström 1998). Therefore, it could also be possible that the HBL genes for strains BC2, BC11, BT2 and BC13 were heterogeneous and thus showed different haemolytic activities on the blood agar plate. For the haemolysis study, we have found that the time required to form the haemolytic zone was not the same for different B. cereus strains.

When the ETF/ETR primers reported by Agata et al. (1995) were used to amplify the bceT genes of B. cereus group strains, 41 of the 84 B. cereus strains, two of the three B. mycoides strains and eight of the nine B. thuringiensis strains generated the expected PCR products with molecular weight equal to 428 bp. Granum et al. (1996) have reported that bceT is not present in five of the seven food-poisoning B. cereus strains. Mäntynen & Lindström (1998) tested 80 strains of Bacillus spp., including 58 B. cereus group cells, and found that only a bceT gene model strain, i.e. strain B-4ac, was positive in amplification of the bceT gene. None of the B. cereus strains they collected showed the expected bceT PCR product. In contrast, our study shows that the ratio for B. cereus strains carrying the bceT gene was much higher, i.e. 49% of the total of 84 B. cereus strains. In addition, 14 (50%) of the 28 food isolates and 17 (57%) of the 30 outbreak-associated strains were bceT PCR-positive.

ENTA and ENTB primers have been used to amplify the entire entFM gene ( Asano et al. 1997 ). The 45 kD enterotoxin (EntFM) isolated from B. cereus strain FM-1 has been shown to be cytotoxic to the Vero cells, although none of the haemolytic activity has been found ( Shinagawa et al. 1991 ). As shown in Table 3, the 1269 bp PCR products amplified from the entFM gene were found for 78 of the 84 B. cereus strains, one of the three B. mycoides strains and seven of the nine B. thuringiensis strains. In addition, 27 of the 28 food isolates and all 30 outbreak-associated strains were entFM PCR-positive. Thus, the entFM gene was found to be the most prevalent enterotoxin gene for B. cereus group cells.

Although it is stated that EntFM is cytotoxic but not haemolytic, some strains, such as strains of enterotoxin profile VIII, show continuous haemolytic patterns ( Table 3). Since haemolytic activity could be due to other factors, such as haemolysins H-I and H-II ( Coolbaugh & Williams 1978), lecithinase or phospholipase C ( Titball 1993), haemolysin II, cereolysin or sphingomyelinase ( Granum 1994), continuous haemolytic patterns for strains of enterotoxin profile VIII could be expected. Since investigation regarding the distribution of the entFM gene in B. cereus group strains has not been reported by other laboratories, our data could not be compared with those of other laboratories.

When CHO cells were used to analyse the cytotoxicity of B. cereus group cells, all the B. cereus group strains, except one B. cereus strain, i.e. BC15, and two B. anthracis strains, showed cytotoxicity to the CHO cells. None of the enterotoxin genes was found for the B. cereus BC15 and B. anthracis strains which showed no cytotoxicity. Our results implied that the CHO cell cytotoxicity assay showed a positive result if any of the three types of enterotoxin genes was present in the B. cereus group cells. Finally, we have to point out that a similar toxigenic profile could be found amongst strains of B. cereus, B. mycoides and B. thuringiensis. For example, the toxigenic properties for B. cereus strains BC8, 19, 20, 25, 26 and BCN12 and B. mycoides strain BMY2 are the same. The toxigenic profile for B. thuringiensis strains BT3–BT9 and B. cereus strains BC1, 3, 5, 9, 10, 14, 24, BCN29, 30, 34, 35, 41 and 42 are the same. Thus, from the viewpoint of food safety, a method for the detection of all B. cereus group cells rather than only the B. cereus spp. is important. As for the virulence profiles for B. cereus strains isolated from food samples and samples associated with food-poisoning outbreaks, a clear difference was not found between these two sources.

Design of novel polymerase chain reaction primers for the detection of the sphingomyelinase gene in Bacillus cereus group cells

Four sphingomyelinase (sph) genes for different B. cereus strains have been sequenced ( Johansen et al. 1988 ; Yamada et al. 1988 ; Gilmore et al. 1989 ; Gavrilenko et al. 1993 ). Tandemly arranged genes have been found to encode phosphatidylcholine-preferring phospholipase C and sphingomyelinase. By comparison of these four sph genes of B. cereus strains with those of non-Bacillus strains, primers Ph1 and Ph2 were designed and tested for their specificity on PCR detection of all the B. cereus group cells collected. Under the PCR conditions as described in Materials and Methods, all the B. cereus group cells tested showed positive results ( Table 3). The molecular weight of the PCR products was 558 bp, as expected. Strains of Bacillus spp. other than B. cereus group cells and strains of non-Bacillus spp. did not generate any false-positive result. Part of the PCR results is shown in Fig. 1. Thus, Ph1/Ph2 primers can be used for the specific detection of B. cereus group cells.

image

Figure 1. Assessment of the specificity of polymerase chain reaction (PCR) primers Ph1/Ph2. (a) PCR results for Bacillus cereus strains. Lanes: 1, 100 bp ladder marker; 2–17, PCR results for B. cereus strains BC1, BC2, BC3, BC4, BC5, BC6, BC7, BC8, BC11, BC12, BC13, BCN1, BCN15, BCN22, BCN35 and BCN58. (b) PCR results amplified from non-B. cereus strains. Lanes: 1, 100 bp ladder marker; 2–17, PCR results for B. cereus strain BC3, Staphylococcus aureus, Yersinia enterocolitica, Listeria monocytogenes, Streptococcus faecalis, Lactobacillus buchner, Lact. delbrueckii ssp. lactis, Proteus vulgaris, Enterobacter aerogenes, Serratia marcescens, Clostridium perfringens, B. coagulans, B. sphaericus, B. licheniformis, B. megaterium and B. circulans

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Detection of Bacillus cereus group cells in food samples

Food samples selected for PCR detection were those subjected to a high risk of contamination by B. cereus group cells. These samples were whole milk, cooked rice, egg and pork purchased from local food markets. When such food samples, e.g. milk, were artificially contaminated with B. cereus cells (strain BC3) and incubated in BHIG broth for 8 h, as few as 100 cells g−1 sample could be detected ( Fig. 2). Similar results could be obtained for other food samples, such as cooked rice, eggs and pork ( Table 4). Samples without target cell inoculation showed negative results as assayed by the PCR method and the conventional method using mannitol-egg yolk-polymyxin agar as the culture medium. These food samples, however, were contaminated by endogenous microflora as evidenced by the PCA counting. For these samples, primers Ph1/Ph2 allowed a detection limit of 100 cfu g−1 sample ( Fig. 2).

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Figure 2. Sensitivity of the detection of Bacillus cereus cells in milk and cooked rice samples. Lanes: 1 and 9, polymerase chain reaction (PCR) results for blank without inoculation of target cells; 8, 100 bp ladder marker; 2–7, PCR results amplified from 100−105 cfu target cells ml−1 whole milk; 10–15, PCR results amplified from 100−105 cfu target cells ml−1 cooked rice

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Table 4.  Sensitivity for the polymerase chain reaction (PCR) detection of the sphingomyelinase gene of Bacillus cereus in food samples after pre-enrichment using Ph1/Ph2 primers
PCR
SamplelContamination evel (cfu g−1) *MYP 0Inoculation level (cfu g−1) N§ × 100N × 101N × 102N × 103N × 104N × 105
  • * 

    Endogenous bacterial numbers were enumerated by total plate count.

  • † 

    B. cereus group cells were enumerated by mannitol-egg yolk-polymyxin (MYP) agar. −, B. cereus cells were not shown on MYP agar.

  • ‡ 

    Conditions for PCR assay were as described in Materials and Methods. + and −, Positive and negative PCR result.

  • § 

    N, Number 1–9.

Whole milk0++++++
Cooked eggN§ × 100++++++
Cooked riceN × 102++++++
Cooked porkN × 103++++++

In conclusion, not only the B. cereus spp. but also the B. cereus group cells are potentially toxigenic. Thus, a method for the detection of all B. cereus group cells in food samples is important. In this report, we have designed a set of novel PCR primers which allows the specific detection of all the B. cereus group cells in food samples.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References

The authors would like to thank the National Science Council, Taipei, Taiwan, ROC. The project numbers for this work are NSC 87–2313-B-005–084 and NSC 88–2313-B-005–006.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References
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