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Abstract

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

Seven emetic toxin-producing strains of Bacillus cereus were examined for toxin production in Skim Milk Medium at incubation temperatures ranging from 10 to 50 °C. Minimum and maximum growth temperatures were found to be 12 and 46 °C, respectively. At 12 and 15 °C, levels of toxin production were significantly higher (P < 0·01) than that observed at 30 °C, while no toxin was produced above 37 °C. Increased levels of sporulation were observed at increased temperatures, and no correlation was found between levels of sporulation and toxin production (R2 = 0·086).


Introduction

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

Bacillus cereus is capable of producing several toxins, including an emetic toxin, a necrotizing enterotoxin, phospholipases and haemolysins. Of the diarrhoeal and emetic food-poisoning toxins that this organism produces, the latter is probably the most dangerous as it has been associated with life-threatening acute conditions such as fulminant liver failure and rhabdomyolysis ( Mahler et al. 1997 ). The ability of a synthetically produced emetic toxin to cause fulminant liver failure has also been demonstrated in laboratory mice ( Yokoyama et al. 1999 ). The toxin is an extremely stable dodecadepsipeptide, which acts as a potassium ionophore ( Mikkola et al. 1999 ) and it is unique amongst enterotoxins in that it is resistant to proteolytic degradation, pH extremes and high temperature, surviving 121 °C for 90 min ( Granum and Lund 1997).

Bacillus cereus emetic food poisoning is mainly associated in western countries with Chinese fried-rice dishes and farinaceous foods such as pasta and noodles ( Kramer and Gilbert 1989). One recent study showed that 2% of pre-cooked rice from take-away restaurants in the UK contained B. cereus at >104 cfu g−1, demonstrating that there are many cases of foods being held at inappropriate storage temperatures ( Nichols et al. 1999 ). Psychrotrophic B. cereus strains are an important cause of spoilage in cold-stored dairy produce, and toxigenic strains are of concern as potential causes of food-borne illness from such products ( Sutherland 1993). Several studies have shown that B. cereus strains may be capable of producing diarrhoeal toxins under reduced temperatures ( Christiansson et al. 1989 ; Fermanian et al. 1997 ). Strains of B. cereus will grow and readily produce emetic toxin in skimmed milk at 30 °C ( Szabo et al. 1991 ; Finlay et al. 1999 ), and toxigenic strains have also been shown to grow at 15 °C ( Johnson et al. 1983 ). However, owing to the lack of a satisfactory toxin assay, no work has been done to determine the maximum temperature range over which B. cereus strains can grow and produce emetic toxin. A convenient, semi-quantitative, MTT dye-based assay for detecting the emetic toxin has recently been published ( Finlay et al. 1999 ). Here, the use of this assay to determine emetic toxin production in relation to growth over a range of temperatures by seven B. cereus strains is reported. The significance of the findings are discussed in the contexts of sporulation and food storage temperatures.

Materials and methods

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

Emetic toxin producing strains

The seven strains studied have previously been shown to produce emetic toxin ( Finlay et al. 1999 ). Six of these strains (F4810/72, F3748/75, F3744/75, F4562/75, F4552/75 and F2427/76) were also shown to cause emesis in monkey feeding tests ( Logan et al. 1979 ). Five strains were isolated in association with emetic food poisoning outbreaks (serovar in parentheses): F4810/72 (1), F3744/75 (1) and F4552/75 (3) from vomit, and F4562/75 (1) and F2549A/76 (5) from Chinese pancakes. Strains F3748/75 (1) and F2427/76 (1) were isolated from faeces.

Strain F4810/72 has been used as a standard strain for emetic toxin production by this group and others ( Hughes et al. 1989 ) and was used here as a positive control. All strains were originally obtained from the Central Public Health Laboratory, Colindale, London, UK and have been maintained in the Logan Bacillus collection since 1978.

Culture conditions

Single colonies selected from streak plates on Nutrient Agar (NA) (Oxoid) (30 °C/24 h) were inoculated into 3 ml Nutrient Broth (NB) (Oxoid). These cultures were then incubated overnight at 30 °C with 200 rev min−1 orbital shaking. Cultures were diluted in NB to give final concentrations of approximately 1 × 103 cfu ml−1, in 50 ml Skim Milk Medium (SMM) (Oxoid) in 500 ml conical flasks. Samples of SMM were brought to the required incubation temperature before inoculation and thereafter were incubated as triplicates with vigorous shaking in water-baths at temperatures of 10, 12 and 15 °C. At higher temperatures (30, 37, 43, 46 and 50 °C), SMM samples were prepared as above but incubated with 200 rev min−1 orbital shaking in an air incubator.

Viable counts, spore counts and toxicity assay

Samples (1 ml) were taken aseptically from each replicate culture after 24 h incubation for temperatures 30, 37, 43, 46 and 50 °C. Samples of 1 ml were taken at intervals of 24 h incubation at 15 °C, and at intervals of 48 h at 12 °C; 100 µl of each 1 ml sample were taken to ascertain viable count (i.e. vegetative bacteria and spores). The remaining 900 µl of each sample was placed in a water-bath at 80 °C for 10 min to kill vegetative bacteria; 100 µl of this heat-treated sample were taken to provide a spore count ( Johnson et al. 1982 ). Serial logarithmic dilutions of each sample were made in NB, and 20 µl volumes were pipetted onto NA and incubated at 30 °C to give counts using the Miles and Misra method. Incubation was halted when cultures were observed to have reached stationary phase.

The remaining 800 µl of heat-treated culture were then centrifuged (4500 g, 40 min, 4 °C) to remove cells and large particulates. Supernatant fluids were decanted and autoclaved (121 °C for 15 min) to sterilize and to denature heat-labile toxins such as B. cereus diarrhoeal toxin, while leaving any emetic toxin undamaged. Supernatant fluids were then tested for cytotoxicity using the MTT metabolic staining assay described previously ( Finlay et al. 1999 ).

Statistical analysis

The results of viable counts, spore counts and reciprocal toxin titres were expressed as log10 mean ± standard error of the mean (derived from triplicate culture of all of the bacterial strains). A paired two-tailed t-test was used to evaluate the statistical significance (P < 0·01) of changes in growth, sporulation or toxicity values with temperature. Analysis of relationship between spore counts and emetic toxin production across the range of growth temperatures was performed by regression analysis (95% confidence interval) of paired values for all bacterial strains.

Results

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

None of the seven strains grew at 10 °C in SMM and therefore, 12 °C was considered to be the lower limit for growth and emetic toxin production. At 12 °C ( Fig. 1), six of the seven strains grew exponentially from day 2, when they gave a viable count of 3·8 (± 0·89), to stationary phase at day 10, when they gave a viable count of 7·45 (± 1·76). Strain F2549A/76 did not grow at 12 °C. Spores were also produced by the six strains at 12 °C but at lower levels than those observed at higher temperatures of incubation (see below), with the maximum mean spore count being 1·60 (± 0·38) on day 4 ( Fig. 1). Five of the six strains produced toxin at 12 °C; one strain (F2427/76), although growing well to a viable count of 8·05 (± 0·48) with a spore count of 2·97 (± 1·56), did not produce detectable emetic toxin at 12 °C. Emetic toxin was detectable after 4 days of incubation at 12 °C, with a mean toxin titre of 0·49 (± 0·12), when the mean viable count had reached 4·40 (± 1·04). This was in contrast to toxin only being detectable at the higher incubation temperature of 15 °C, when viable counts had reached at least 6·0 (± 0·94). The highest mean toxin titre achieved by the five producing strains at 12 °C was 4·02 (± 0·95) at day 12 ( Fig. 1). This was a significant (P < 0·01) increase over the highest toxin titre of 3·50 (± 1·00) obtained at 30 °C.

image

Figure 1. Mean log10 count and emetic toxin production by six strains of Bacillus cereus at 12 °C over 12 days. (▪), Viable count; (□), spore count; (▵), toxin titre

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All seven strains grew rapidly at 15 °C ( Fig. 2), reaching a mean viable count of 7·42 (± 1·75) by day 3 and entering stationary phase by day 5. Spores were detectable after 48 h of incubation and ranged from a mean spore count of 1·47 (± 0·35) to a mean spore count of 3·00 (± 0·71) from day 2 to 5 ( Fig. 2). Emetic toxin was produced by all seven strains and was first detectable at day 2 for strains F3744/75 and F4562/75, and day 3 for strains F4810/72, F3748/78, F2427/76, F4552/75 and F2549A/76, reaching a maximum mean toxin titre of 4·15 (± 0·97) by day 6 ( Fig. 2).

image

Figure 2. Mean log10 count and emetic toxin production by seven strains of Bacillus cereus at 15 °C over 7 days. (▪), Viable count; (□), spore count; (▵), toxin titre

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At 30 °C and 37 °C, all seven strains had grown to stationary phase by 24 h of incubation ( Fig. 3), with the mean viable counts being 8·62 (± 0·09) and 8·6 (± 0·05), respectively. Large numbers of spores were also found, with mean spore counts of 4·83 (± 0·47) and 6·2 (± 0·84), respectively ( Fig. 3). All strains produced toxin at 30 °C ( Fig. 3) with a mean titre of 3·50 (± 1·00), but only four strains (F4810/72, F3748/78, F4562/75 and F4552/75) produced emetic toxin at 37 °C ( Fig. 3), with a mean titre of 1·63 (± 1·33).

image

Figure 3. Mean log10 count for seven strains of Bacillus cereus, and toxin titre for producing strains, after 24 h, at 30, 37, 43 and 46 °C. (▪), Viable count; (□), spore count; (▵), toxin titre

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No toxin was produced at incubation temperatures above 37 °C, even when strains reached stationary phase within 24 h, and spore counts were significantly (P < 0·01) higher at 37, 43 and 46 °C than at 12 or 15 °C. For example, at 43 °C ( Fig. 3) all strains grew, giving a mean viable count of 7·8 (± 0·09) and all produced spores with a mean count of 2·9 (± 0·76). At 46 °C ( Fig. 3) only four strains (F4810/72, F3744/75, F3748/78 and F2427/76) grew, giving a mean viable count of 7·9 (± 0·13) and large numbers of spores (mean count 4·2 ± 0·87). Regression analysis (95% confidence interval) of paired values for all bacterial strains across all temperatures tested showed no correlation (R2 = 0·086) between spore production and emetic toxin production. The upper limit for growth of any strain was 46 °C, as none grew at 50 °C.

Discussion

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

Te Giffel et al. (1997) have shown that 40% of skim milk samples in the Netherlands may contain mesophilic B. cereus counts of between 50 and 5000 cfu ml−1, with 73% of their isolates shown to produce diarrhoeal toxin by western immunoblot. As 53% of B. cereus isolated in that study were capable of growth at 7 °C, and psychrotrophic strains of B. cereus have been shown to be present in several other studies of milk ( Christiansson et al. 1989 ; Dufrenne et al. 1994 ; Sutherland and Murdoch 1994; Larsen and Jørgensen 1998), it was considered possible that some B. cereus isolates might produce emetic toxin at low temperatures. Also, van Netten et al. (1990) described an outbreak of nausea and vomiting in the Netherlands which was associated with B. cereus in pasteurized milk. Strains isolated from that outbreak were shown to grow at 4 °C but were not tested for emetic toxin production.

In the present work, the lower temperature limit for growth and emetic toxin production was found to be 12 °C, with emetic toxin being produced by five strains at 12 °C and detectable after 4 days of growth. Interestingly, emetic toxin was produced in much greater quantities at both 12 and 15 °C than at any of the higher temperatures tested. In parallel with this, the lowest levels of growth (mean viable count of 4·40 ± 1·04) capable of giving detectable emetic toxin occurred at 12 °C. At 15 °C, toxin production was not detectable until the viable count reached 6·00 (± 0·94), and Agata et al. (1996) have also shown that at 30 °C, toxin production in SMM only became detectable once the viable count had reached a level greater than 6·0. The standard temperature used by this group ( Finlay et al. 1999 ) and others ( Hughes et al. 1989 ; Szabo et al. 1991 ) for emetic toxin production in SMM was 30 °C. It is now clear that for many strains, incubation at 12 or 15 °C will allow development of significantly (both P < 0·01) higher levels of toxin production than will 30 °C, albeit over longer incubation periods. This may be of value, for example, in preparing toxin for purification studies. From an applied point of view, these results are most important. Bacillus cereus strains which produce emetic toxin have reportedly been recovered from dairy products ( Beattie and Williams 1999). Therefore, substantial toxin levels may be obtained in these products if even moderate temperature abuse (12 °C− 15 °C) is allowed to occur for periods of several days. This would be particularly important for highly flavoured products in which off-flavours and smells associated with spoilage might not be detected, as noted by Sutherland and Murdoch (1994). The results may be equally important for other food types, but the influence of other food constituents remains to be determined.

The upper temperature limit for emetic toxin production was found to be 37 °C, while the maximum temperature for growth by any strain was 46 °C. Fermanian et al. (1994) have also shown that in eight ‘potentially pathogenic’ strains (including one emetic outbreak strain), no growth was observed in Brain-Heart Infusion medium above 46 °C. The data presented here support these findings. Lack of detectable toxin production above 37 °C implies that cooked foods held for long periods at raised temperatures are unlikely to be at risk of emetic toxin development. Also, toxin production at 37 °C was greatly reduced compared with that at lower temperatures, which implies that emetic toxin production in the gastro-intestinal tract is likely to be minimal. As toxin was produced in greatest amounts at 12 and 15 °C and was not produced at 43 or 46 °C, when high viable counts were obtained, it is clear that emetic toxin production is regulated by temperature. Whether regulation occurs at the transcriptional, translational or post-translational level remains to be determined. The data presented here suggest that emetic toxin production below 12 °C and above 37 °C is unlikely.

Toxin production is known to be associated with sporulation in Clostridium perfringens ( Duncan et al. 1972 ). Andersson et al. (1998) found that growth of emetic toxin-producing B. cereus on tryptic soy plates for 10 days at 28 °C gave mostly sporulated and lysed cells, yielding 0·5 ng purified emetic toxin per 104 cfu B. cereus. They also showed that toxic cooked-rice samples contained mainly sporulated and lysed cells, with very few vegetative cells. These findings suggested that B. cereus emetic toxin production might be associated with sporulation. In this study, the presence of spores was transient (often appearing, disappearing then reappearing as incubation progressed) in all strains at 12 and 15 °C, and there was no correlation between heat-resistant spore production and emetic toxin. As large numbers of spores can be produced in the absence of detectable emetic toxin, this suggests that the toxin is not a spore component.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References
  • Agata, N., Ohta, M., Mori, M. (1996) Production of an emetic toxin, Cereulide, is associated with a specific class of Bacillus cereus. Current Microbiology 33, 67 69.
  • Andersson, M.A., Mikkola, R., Helin, J., Andersson, M.C., Salkinoja-Salonen, M. (1998) A novel sensitive bioassay for detection of Bacillus cereus emetic toxin and related depsipeptide ionophores. Applied and Environmental Microbiology 64, 1338 1343.
  • Beattie, S.H. & Williams, A.G. (1999) Detection of toxigenic strains of Bacillus cereus and other Bacillus sp. with an improved cytotoxicity assay. Letters in Applied Microbiology 28, 221 225.
  • Christiansson, A., Naidu, A.S., Nilsson, I., Wadström, T., Petterson, H.E. (1989) Toxin production by Bacillus cereus dairy isolates in milk at low temperatures. Applied and Environmental Microbiology 55, 2595 2600.
  • Dufrenne, J., Soentore, P., Tatini, S., Day, T., Notermans, S. (1994) Characteristics of Bacillus cereus related to safe food production. International Journal of Food Microbiology 23, 99 109.
  • Duncan, C.L., Strong, D.H., Sebald, M. (1972) Sporulation and enterotoxin production by mutants of Clostridium perfringens. Journal of Bacteriology 110, 378 391.
  • Fermanian, C., Fremy, J.M., Claisse, M. (1994) Effect of temperature on the vegetative growth of type and field strains of Bacillus cereus. Letters in Applied Microbiology 19, 414 418.
  • Fermanian, C., Lapeyre, C., Fremy, J.M., Claisse, M. (1997) Diarrhoeal toxin production at low temperature by selected strains of Bacillus cereus. Journal of Dairy Research 64, 551 559.
  • Finlay, W.J.J., Logan, N.A., Sutherland, A.D. (1999) Semi-automated metabolic staining assay for Bacillus cereus emetic toxin. Applied and Environmental Microbiology 65, 1811 1812.
  • Granum, P.E. & Lund, T. (1997) Bacillus cereus and its food poisoning toxins. FEMS Microbiology Letters 157, 223 228.DOI: 10.1016/s0378-1097(97)00438-2
  • Hughes, S., Bartholomew, B., Hardy, J.C., Kramer, J.M. (1989) Potential application of a Hep-2 cell assay in the investigation of Bacillus cereus emetic-syndrome food poisoning. FEMS Microbiology Letters 52, 7 12.
  • Johnson, K.M., Nelson, C.L., Busta, F.F. (1982) Germination and heat resistance of Bacillus cereus spores from strains associated with diarrhoeal and emetic food-borne illnesses. Journal of Food Science 47, 1268 1271.
  • Johnson, K.M., Nelson, C.L., Busta, F.F. (1983) Influence of temperature on germination and growth of spores of emetic and diarrhoeal strains of Bacillus cereus in a broth medium and in rice. Journal of Food Science 48, 286 287.
  • Kramer, J.M. & Gilbert, R.J. (1989) Bacillus cereus and other Bacillus species. In Foodborne Bacterial Pathogens ed. Doyle, M.P. pp. 21 70. New York: Marcel Dekker.
  • Larsen, H.D. & Jørgensen, K. (1998) Growth of Bacillus cereus in pasteurized milk products. International Journal of Food Microbiology 46, 173 176.DOI: 10.1016/s0168-1605(98)00188-3
  • Logan, N.A., Capel, B.J., Melling, J., Berkeley, R.C.W. (1979) Distinction between emetic and other strains of Bacillus cereus using the API system and numerical methods. FEMS Microbiology Letters 5, 373 375.
  • Mahler, H., Pasi, A., Kramer, J.A. et al. (1997) Fulminant liver failure in association with the emetic toxin of Bacillus cereus. New England Journal of Medicine 336, 1142 1148.
  • Mikkola, R., Saris, N.E.L., Grigoriev, P.A., Andersson, M.A., Salkinoja-Salonen, M.S. (1999) Ionophoretic properties and mitochondrial effects of cereulide – The emetic toxin of Bacillus cereus. European Journal of Biochemistry 263, 112 117.DOI: 10.1046/j.1432-1327.1999.00476.x
  • Nichols, G.L., Little, C.L., Mithani, V., DeLouvois, J. (1999) The microbiological quality of cooked rice from restaurants and take-away premises in the United Kingdom. Journal of Food Protection 62, 877 882.
  • Sutherland, A.D. (1993) Toxin production by Bacillus cereus in dairy products. Journal of Dairy Research 60, 569 574.
  • Sutherland, A.D. & Murdoch, R. (1994) Seasonal occurrence of psychrotrophic Bacillus species in raw milk, and studies on the interactions with mesophilic Bacillus sp. International Journal of Food Microbiology 21, 279 292.
  • Szabo, R.A., Spiers, J.I., Akhtar, M. (1991) Cell culture detection and conditions for production of a Bacillus cereus heat-stable toxin. Journal of Food Protection 54, 272 276.
  • Te Giffel, M.C., Beumer, R.R., Granum, P.E., Rombouts, F.M. (1997) Isolation and characterization of Bacillus cereus from pasteurized milk in household refrigerators in the Netherlands. International Journal of Food Microbiology 34, 307 318.DOI: 10.1016/s0168-1605(96)01204-4
  • Van Netten, P., Vand De Moosdijk, A., Van Hoensel, P., Mossel, D.A.A., Perales, I. (1990) Psychrotrophic strains of Bacillus cereus producing enterotoxin. Journal of Applied Bacteriology 69, 73 79.
  • Yokoyama, K., Ito, M., Agata, N. et al. (1999) Pathological effect of cereulide, an emetic toxin of Bacillus cereus, is reversible in mice . FEMS Immunology and Medical Microbiology 24, 115 120.DOI: 10.1016/s0928-8244(99)00017-6