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Bacillus cereus emetic toxin production in relation to dissolved oxygen tension and sporulation
Food Microbiology, 2002, 19, 423^430 Available online at http://www.idealibrary.com on
doi:10.1006/yfmic.504
ORIGINAL ARTICLE
Bacillus cereus emetic toxin production in relation to dissolved oxygen tension and sporulation W. J. J. Finlay1;2, N. A. Logan1 and A. D. Sutherland1;
Seven emetic toxin-producing strains of Bacillus cereus were examined for growth, sporulation and toxin production in skim milk medium in shaken and unshaken batch cultures under aerobic, microaerobic and anaerobic atmospheres. High levels of toxin production were detected in aerobic and microaerobic cultures. Static cultures yielded 90% less toxin than equivalent shaken cultures. Emetic toxin production was detected in aerobic and microaerobic cultures when bacterial counts reached 4 6?0 log10 cfu ml 1, which also coincided with spore production. However, no correlation was found between spore count and toxin production (R2 =0?032). In anaerobic culture, production of emetic toxin was undetectable and the dissolved oxygen tension in the growth medium fell below 125746 ppm within 6 h of inoculation. In aerobic culture, most toxin was produced between 16 and 22 h of incubation, when the dissolved oxygen tension had decreased to 60 70 ppm.Therefore, while O2-dependent respiration is fundamentally required for production of the emetic toxin, most toxin production occurs when little free oxygen is available. # 2002 Elsevier Science Ltd. All rights reserved.
Introduction Bacillus cereus is capable of producing several toxins, including a necrotizing enterotoxin, an emetic toxin, phospholipases, proteases and haemolysins. Of the food-poisoning toxins, the emetic toxin 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). Fulminant liver failure has also been shown to be inducible in laboratory mice using synthetically produced emetic toxin (Yokoyama et al. 1999). The emetic toxin is an extremely stable * Corresponding author. Fax: +44 141 331 3208; E-mail: [email protected] 0740 -0020/02/050423+08 $35.00/0
dodecadepsipeptide, which acts as a potassium ionophore (Mikkola et al. 1999), and which is resistant to proteolytic degradation, pH extremes and high temperature, surviving 1211C for 90 min (Granum and Lund 1997). In western countries, B. cereus emetic food poisoning is associated mainly with Chinese fried rice dishes and farinaceous foods such as pasta and noodles. One recent study showed that 2% of pre cooked rice from take-away restaurants in the UK contained B. cereus at 4104 cfu g 1 (Nichols et al. 1999). B. cereus is also associated with dairy produce; Te Gi¡el et al. (1997) showed that 40% of the skim milk samples they collected in the Netherlands contained mesophilic B. cereus at levels between 50 and 5000 cfu ml 1. r 2002 Elsevier Science Ltd. All rights reserved.
Received: 9 April 2002 1 School of Biological and Biomedical Sciences, Glasgow Caledonian University, Cowcaddens Road, Glasgow G4 OBA, Scotland, UK 2 Current address: Centre for Biologics Evaluation and Research, US Food and Drug Administration, 29 Lincoln Drive, Bethesda, Maryland 20892, USA
424 W. J. J. Finlay et al.
The minimum amino acid nutritional requirements for emetic toxin production have been described by Agata et al. (1999), and we have recently reported the ability of B. cereus strains to produce large amounts of emetic toxin in skim milk medium (SMM) under lowered growth temperatures (Finlay et al. 2000). However, other fundamental environmental requirements for production of the emetic toxin have not yet been investigated fully. Several researchers (Hughes et al. 1989, Szabo et al. 1991, Agata et al. 1996) have shown that high levels of emetic toxin production in liquid cultures require vigorous shaking (up to a maximum of 200 rpm). This suggested that oxygenation of the culture medium in vitro may be an important stimulus for emetic toxin production. If this is the case, then oxygen tension in the microenvironment may also be of critical importance in production of the emetic toxin in foods. Enterotoxin production is known to be associated with the formation of spores in other genera such as Clostridium (Duncan et al. 1972, Labbe and Duncan 1977, Ryu and Labbe 1989). Andersson et al. (1998) also found that growth of emetic toxin-producing B. cereus strains on tryptic soy agar plates for 10 days at 281C created mostly sporulated and lysed cells and yielded 0?5 ng of puri¢ed emetic toxin per 104 cfu of B. cereus. They also showed that cooked rice samples, in which emetic toxin had been produced, contained mainly sporulated and lysed cells with very few vegetative cells. These ¢ndings suggested that production of the B. cereus emetic toxin might also be associated with sporulation. To investigate these factors, the growth, sporulation, oxygen consumption and toxin production of seven known emetic B. cereus strains were examined in static and shaken batch cultures under aerobic, microaerobic and anaerobic conditions.
Materials and Methods Bacterial strains The seven strains studied have previously been shown to produce emetic toxin (Finlay et al. 1999, 2000). Six of the strains (F4810/72, F3748/
75, F3744/75, F4562/75, F4552/75, and F2427/76) have also been 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 obtained originally from the Central Public Health Laboratory, Colindale, London, UK and have been maintained in the Logan Bacillus collection, now at Glasgow Caledonian University, since 1978.
Culture conditions Single colonies of each strain tested were selected from streak plates on Nutrient Agar (NA) (Oxoid, UK) (301C/overnight) and inoculated into 3 ml Nutrient Broth (NB) (Oxoid, UK). These cultures were then incubated overnight at 301C with 200 rpm orbital shaking. Cultures were diluted in NB to give ¢nal concentrations of approximately 1 103 cfu ml 1 in triplicate 50 ml volumes of SMM (Oxoid, UK) in 500 ml conical £asks. Skim milk medium has been shown to be the optimum medium for emetic toxin production (Szabo et al. 1991, Finlay et al. 1999). These cultures were placed immediately in aerobic (ambient O2 ), microaerophilic or anaerobic environments. Modi¢ed oxygen conditions were created in Gas Pak jars with a microaerobic catalyst sachet or anaerobic catalyst sachet to produce 410% O2 and 0% O2, respectively (all BBL Microbiology Systems, UK), with the exception that static anaerobic cultures were incubated under 0% O2 in an anaerobic cabinet (Don Whitley Ltd, UK). Anaerobic conditions were con¢rmed using indicator strips (Oxoid, UK). Triplicate cultures of each strain under aerobic, microaerobic and anaerobic conditions were incubated at 301C for 24 h with or without shaking (200 rpm).
B. cereus emetic toxin 425
Continuous measurement of oxygen tension in stirred batch cultures Probes for the measurement of dissolved oxygen tension (DOT) are ine¡ective in static liquids owing to the depletion of O2 in the immediate vicinity of the probe by the electrode itself. As measurements of DOT in static cultures would therefore be misleading, DOT was only measured in agitated (stirred) cultures; it was measured in parts per million (ppm). Cultures of strain F4810/72 were prepared as described above but with the addition of a sterile 30 mm rod-shaped magnetic stirrer. Vigorous magnetic stirring was applied to the culture to encourage oxygenation of the medium and so mimic the rapid shaking conditions described by other authors (Hughes et al. 1989, Szabo et al. 1991, Agata et al. 1996).This allowed the e¡ective use of a ‘Micro O2 probe’ (Lazar Research Laboratories Inc., USA), to monitor DOT in the culture continuously. A Gas Pak (BBL) jar was modi¢ed to allow DOT measurement; a small hole, su⁄cient to accommodate the electrical wire extending from the O2 probe, was drilled in the lid of the jar. This hole was then sealed on both sides using epoxy resin; to stabilize the O2 probe and to ensure an airtight seal. An uninoculated negative control culture (to measure normal DOT levels in SMM at 301C over the incubation period) was created using SMM with the addition of 20 mg ml 1 Gentamycin (Sigma, UK), to inhibit the growth of any bacterial contaminants. Digital DOT readings were taken regularly, and DOT was also recorded continually on a chart recorder (Linseis, UK), for 24 h with constant stirring under aerobic, microaerobic or anaerobic conditions. Experiments using the di¡erent growth conditions were performed on three separate occasions. All cultures from DOT experiments were analysed for bacterial growth and toxin production as described below.
Total counts, spore counts and toxicity assay Two 1 ml samples were taken aseptically from each batch culture after 24 h of incubation. One sample was placed in a water bath at 801C for 10 min in order to kill vegetative cells of B.
cereus and provide a presumptive count for spores (Johnson et al. 1982).The other 1 ml sample was unheated, and used to provide a viable count. Log serial dilutions were prepared in NB and viable or spore counts were estimated by plating duplicate 20 ml drops of each dilution on NA. The remainder of the culture was then centrifuged (4500 g 40 min 1 41C 1 ) to remove bacteria and large particulates. Supernatants were decanted and autoclaved (1211C for 15 min) to sterilize and to denature heat-labile toxins such as B. cereus diarrhoeal toxin. Supernatants were then tested for the emetic toxin using the MTT metabolic staining assay described previously (Finlay et al. 1999).
Growth curve of aerobic culture A growth curve for shaken aerobic culture was also performed using triplicate cultures of strain F4810/72, prepared as above. One-millilitre samples were removed aseptically every 2 h, up to 24 h of incubation. Two 100 ml aliquots were removed from this sample and used as described above to provide spore and viable counts. The remaining 800 ml of sample was then used to prepare supernatant for toxicity testing, as described above.
Statistical analysis The results of viable counts and spore counts (derived from triplicate cultures of all of the bacterial strains) were expressed as log10 mean7standard deviation. Toxin titres of the individual strains were expressed as means of their reciprocal toxin titres, as described previously (Finlay et al. 1999). Grand mean reciprocal toxin titre7grand mean standard deviation values (to show overall variability) were also calculated for triplicate cultures of all strains. A paired two-tailed t-test was used to evaluate the statistical signi¢cance of changes in growth, sporulation or toxicity values with change in atmospheric conditions. Analysis of the relationship between spore counts and emetic toxin production across the range of atmospheric conditions was performed by regression analysis of paired values
426 W. J. J. Finlay et al.
(95% con¢dence interval) for all bacterial strains.
Results The results from triplicate cultures of each of the seven strains, when tested under the same conditions, were very similar and data are, therefore, presented as mean values. Shaken aerobic and microaerobic conditions gave similar mean viable counts for all strains at 24 h (8?670?3 and 8?570?2 log10 cfu ml 1 respectively) (Table 1). Static aerobic and microaerobic growth also yielded similar mean viable counts at 24 h (8?270?5 and 7?870?8 log10 cfu ml 1 , respectively) (Table 1). However, all strains grew less abundantly under shaken and static anaerobic conditions, producing mean viable counts which were signi¢cantly (Po0?01) lower than equivalent cultures in aerobic or microaerobic conditions (7?470?2 and 7?370?2 log10 cfu ml 1 , respectively) (Table 1). No signi¢cant rise in total count was observed under any of the atmospheric conditions when the cultures were shaken (P40?05) (Table 1). Toxin was detectable in shaken and static aerobic and microaerobic cultures but not in static or shaken anaerobic cultures. This was despite mean viable counts in anaerobic culture being consistently 46?0 log10 cfu ml 1 , which is su⁄cient to produce detectable toxin in shaken aerobic culture (Hughes et al. 1989, Agata et al. 1996, Finlay et al. 2000). Mean spore counts for all strains under all conditions were typically 3 orders of magnitude lower than their respective mean viable
counts (Table 1). Shaken anaerobic conditions produced spore counts similar to those observed in shaken aerobic growth (4?870?9 and 4?870?5 log10 cfu ml 1 , respectively). Shaken microaerobic growth however, gave signi¢cantly (Po0?01) increased mean spore counts (6?770?3 log10 cfu ml 1 ) (Table 1), without leading to signi¢cantly (P40?05) higher toxicity than that produced in shaken aerobic growth (Table 1). Static aerobic and microaerobic conditions gave respective spore counts of 5?170?1 and 5?270?4, which were not signi¢cantly (P40?05) di¡erent from those observed in shaken aerobic culture (Table 1). Despite this, toxin levels dropped by 89% and 87%, respectively, in static aerobic and microaerobic cultures, when compared to shaken cultures (Table 1). Spore counts in static anaerobic culture were not signi¢cantly di¡erent (P40?05) from those observed in static aerobic and microaerobic culture (Table 1). Regression analysis of spore counts and emetic toxin production across the range of atmospheric conditions, therefore, found no correlation between sporulation level and toxin production (R2 = 0?032). However, when a growth-curve analysis for strain F4810/72 was performed, it showed that emetic toxin production in aerobic culture became detectable after 6 h of incubation, which coincided with spores becoming evident (mean spore count of 2?470?4 log10 cfu ml 1 ) (Fig. 1). All strains produced high levels of toxin under both aerobic and microaerobic conditions (Table 1). Mean toxin titres from shaken microaerobic cultures were 20% lower than those obtained from shaken aerobic cultures. In comparison with static cultures, shaken cultures
Table 1. Mean log10 viable count, spore count and reciprocal toxin titre for seven strains of Bacillus cereus under varying atmospheric conditions Culture conditions Aerobic shaken Aerobic static Microaerobic shaken Microaerobic static Anaerobic shaken Anaerobic static
Total log10 cfu ml 8?6 (70.3)* 8?2 (70.5) 8?5 (70?2) 7?8 (70?8) 7?4 (70?2) 7?3 (70?2)
1
Spore log10 cfu ml 4?8 (70.5) 5?3 (70?1) 6?7 (70?3) 5?2 (70?4) 4?8 (70?4) 4?8 (70?9)
1
Reciprocal toxin titre 3191 (710) 358 (7138) 2547 (7781) 342 (7102) 0 0
*Error values in parentheses are standard deviation for triplicate cultures of all the seven strains analysed (i.e. n = 21).
B. cereus emetic toxin 427
viable count
spore count
toxin titre
log10 count ml-1
8
2000
7 6
1500
5
1000
4 500
3 2
Reciprocal toxin titre
2500
9
0 2
4
6
8 10 12 14 16 18 20 22 24 Time (hours)
Figure 1. Mean (7s.e.) viable count, spore count and reciprocal toxin titres for B. cereus strain F4810/72 at 301C over 24 h, under aerobic growth conditions.
gave signi¢cant (Po0?01) increases in toxicity under both microaerobic and aerobic conditions (Table 1). Microaerobic cultures showed less consistency in toxin titres following shaken incubation (Table 1). None of the strains produced toxin under either static or shaken anaerobic conditions (Table 1). The modi¢ed Gas Pak jar remained airtight when anaerobic atmospheric conditions were induced, as evidenced by a DOT of o20 ppm and by the positive reaction of anaerobic indicator strips. Also, mean viable counts, spore counts and toxicity values were not signi¢cantly di¡erent (P40?05) from those observed in unmodi¢ed gas jars. All cultures were found to have initial DOT values of 762724 ppm at 301C. This is in agreement with the normal DOT in distilled water of 760 ppm at 301C and at atmospheric pressure of 750 mm Hg. In the negative control stirred aerobic culture, DOT was maintained at a constant level over 24 h, with an initial value of 759714 ppm and a ¢nal value of 761717 ppm.Therefore, the observed reduction in DOT values below 760 ppm in aerobic cultures (Fig. 2) was considered to re£ect oxygen consumption by bacterial growth. In aerobic culture, DOT dropped slowly to 710712 ppm during the ¢rst 4 h of incubation and then decreased more rapidly to 2767 21ppm after 8 h of incubation reaching its minimum value of 68711ppm at 22 h. In microaerobic culture, the catalyst sachet brought about a rapid reduction of DOT to 461727 ppm after 2 h, and a slower reduction to 183745 ppm at
8 h; DOT then continued to drop steadily, reaching 6479 ppm after 16 h of incubation and 57713 ppm after 24 h of incubation. In anaerobic cultures, the catalyst sachet brought about a rapid reduction of DOT to 348742 ppm in 2 h. Reduction in DOT then slowed, reached 2578 ppm after 8 h, and maintained this level until 24 h of culture, when DOT was 16711ppm. Aerobic growth-curve data (Fig. 1) showed that toxin production became detectable (reciprocal toxin titer 574) after 6 h of incubation, when the viable count had reached 6?170?1. At this time, DOT values for the aerobic, microaerobic and anaerobic cultures were 561713, 278741 and 125746 ppm, respectively (Fig. 2). In aerobic growth, levels of emetic toxin rose to a mean reciprocal toxin titre of 6827295 after 18 h of incubation, by which point cultures had entered stationary phase (Fig. 1) and DOT had declined to 72726 (Fig. 3). Toxin production in aerobic culture then rose considerably, with no observed increase in DOT, reaching a maximum level of 204870 after 22 h of incubation (Fig. 3).
Discussion Labbe and Duncan (1977) demonstrated that enterotoxin production in Clostridium perfringens occurs coincidentally with sporulation. Shinagawa et al. (1992) have also suggested that B. cereus emetic toxin is produced only when sporulation is underway, being detected 5 h after sporulation was ¢rst observed. In the present study, growth-curve analysis of strain F4810/72 in SMM also revealed that emetic toxin became detectable as spore production began. However, the spore counts evident when emetic toxin production became detectable (2?470?4 log10 cfu ml 1 ) were more than 2?0 log10 lower than those associated (5?0 log10 cfu ml 1 ) with clostridial enterotoxin production (Labbe and Duncan 1977), and more than 4 log lower than the ¢gure of 6?7 log10 cfu ml 1 reported to be required for B. cereus emetic toxin production in rice broth culture (Shinagawa et al. 1992). Spore counts observed in this study were generally more than 3 log10 lower than the viable count levels.
428 W. J. J. Finlay et al.
Dissolved Oxygen Tension (log10 ppm)
8 7 6 5 4 3 2 1 0 2
4
6
8
10
12
14
16
18
20
22
24
TIME (Hours)
Figure 2. DOT measurement aerobic (
), microaerobic ( ) and anaerobic growth conditions, for B. cereus strain F4810/72, in stirred culture at 301C over 24 h. 8
2250
8.5
2000
8.0
6
1750
7.5
5
1500
(
)
1000 3 750 2
7.0 6.5 6.0 5.5
log10 viable count
1250 4
Reciprocal Toxin Titre
Dissolved Oxygen Tension (log10 ppm)
7
5 500 4.5
1 250 0
4.0
0 2
4
6
8
10
12
14
16
18
20
22
24
TIME (Hours)
Figure 3. DOT measurements under aerobic growth conditions ( ), versus mean growth (B) and emetic toxin production (&) under aerobic conditions, for B. cereus strain F4810/72, in stirred culture at 301C over 24 h.
In shaken microaerobic growth, sporulation occurred at a higher level than in aerobic growth, without a corresponding increase in toxin production in relation to aerobic culture. Static aerobic or microaerobic culture also produced spore levels comparable to those
achieved in shaken aerobic growth, but toxin levels were almost 90% lower than those produced by shaken culture. Also, both shaken and static anaerobic cultures yielded similar numbers of spores to those achieved by shaken aerobic cultures. Regression analysis thus
B. cereus emetic toxin 429
showed no correlation (R2 = 0?032) between levels of sporulation and emetic toxin production in the di¡erent cultures. As no toxin was detected in any anaerobic culture, these data support our earlier ¢ndings that B. cereus spore formation can proceed without emetic toxin production (Finlay et al. 2000). It also supports our suggestion that the emetic toxin, while becoming detectable coincidentally with spore development, is not produced in direct association with the sporulation process. Production of B. cereus enterotoxin has been shown to proceed under anaerobic growth conditions (Glatz and Goepfert 1976), but the present study shows that the emetic toxin is not produced under anaerobic conditions. These ¢ndings, therefore, suggest that production of the emetic toxin is unlikely to be regulated by the same mechanism as the B. cereus enterotoxins. Analysis of the growth curve of strain F4810/ 72 under aerobic culture conditions showed that emetic toxin production only became detectable when viable counts reached 6?0 log10 cfu ml 1, at 6 h of incubation. This is consistent with the ¢ndings of Hughes et al. (1989) and Agata et al. (1996), who showed that emetic toxin production begins in SMM after 6^8 h shaken incubation at 301C, at a viable count of roughly 6?0 log10 cfu ml 1. It has also recently been reported that toxin production occurs at 151C in SMM, when viable count reaches 6?0 log10 cfu ml 1 or above (Finlay et al. 2000). These ¢ndings suggest that expression of the emetic toxin is either regulated by cell density, or that toxin concentration only reaches a detectable level when counts above 6?0 log10 cfu ml 1 are achieved. Glatz and Goepfert (1976) have shown that, in the growth of B. cereus in 300 ml cultures performed in fermenter vessels (with agitation), DOT drops rapidly during exponential phase growth, and conditions become anaerobic once cultures enter stationary phase. As B. cereus enters late exponential phase O2 demand increases, rendering maintenance of a high DOT level di⁄cult (Spira and Silverman 1979), even with rapid agitation and sparging of the medium with O2 (Bauer and Ziv 1976). The DOT curve observed in the present study, for growth in agitated aerobic culture, shows that rapid O2 consumption also occurs in small-
scale batch (50 ml) culture and that in late exponential and stationary phase growth, low levels of DOT occur (72726 ppm). Rates of O2 consumption in milk for B. cereus in the present study were similar to those observed for Escherichia coli by Goldberg et al. (1994); in aerobic and microaerobic fermenter cultures, rapid reduction of DOT occurred during exponential phase growth and the culture became anaerobic once the population of E. coli achieved a viable count of 6?0 log10 cfu ml 1 . Measurement of DOT in stirred cultures revealed that toxin production could be detected after 6 h of incubation, when oxygen was still freely available in both aerobic and microaerobic cultures (561713 and 278741ppm, respectively), while in anaerobic culture DOT had dropped to 125746 ppm by this time. However, the majority of toxin was produced between 16 and 22 h of incubation, when aerobic and microaerobic cultures had entered stationary phase and DOT was only 60^70 ppm. Culture in the presence of an anaerobic catalyst sachet reduced DOT to 25 ppm within 8 h, meaning that little or no free oxygen remained in the medium, precluding production of the toxin as the organisms had entered a state of anaerobic respiration. The low DOT that was observed in the late log/stationary phases of growth in cultures performed under aerobic and microaerobic conditions (Fig. 3) was almost certainly caused by bacterial oxygen consumption outstripping the oxygen input achieved by agitation. The amount of oxygen being provided by agitation was, however, su⁄cient to allow aerobic respiration of the cultures (and subsequent emetic toxin production), as even at sub-microaerobic DOT, the di¡usion of O2 into the cytoplasm of bacteria occurs at a rate su⁄cient to allow catabolism (Arras et al. 1998). The ¢ndings presented in this study show that static microaerobic cultures produce comparable amounts of emetic toxin to static aerobic cultures, and that shaken microaerobic and aerobic cultures also produce very similar toxin levels. Free oxygen levels are very low in late log phase and stationary phase shaken cultures, even with rapid agitation of the culture medium, and toxin production in static cultures is 90% lower than that observed in shaken cultures. This, therefore, suggests that
430 W. J. J. Finlay et al.
shaking maintains the minimal levels of DOT required to produce the emetic toxin. Static culture does not allow e⁄cient O2 distribution in the growth medium and this leads to a lower level of toxin production. The ¢ndings reported here imply that while O2 -dependent respiration is required fundamentally for production of the emetic toxin, most toxin production occurs when little free oxygen is available in the growth medium.
References Agata, N., Ohta, M. and Mori, M. (1996) Production of an emetic toxin, Cereulide, is associated with a speci¢c class of Bacillus cereus. Curr. Microbiol. 33, 67^69. Agata, N., Ohta, M., Mori, M. and Shibayama, K. (1999) Growth conditions of and emetic toxin production by Bacillus cereus in a de¢ned medium with amino acids. Microbiol. Immunol. 43, 15^18. Andersson, M. A., Mikkola, R., Helin, J., Andersson, M. C. and Salkinoja-Salonen, M. (1998) A novel sensitive bioassay for detection of Bacillus cereus emetic toxin and related depsipeptide ionophores. Appl. Environ. Microbiol. 64, 1338^1343. Arras, T., Schirawski, J. and Unden, G. (1998) Availability of O2 as a substrate in the cytoplasm of bacteria under aerobic and microaerobic conditions. J. Bacteriol 180, 2133^2136. Bauer, S. and Ziv, E. (1976) Dense growth of aerobic bacteria in a bench-scale fermentor. Biotechnol. Bioeng. 18, 81^94. Duncan, C. L., Strong, D. H. and Sebald, M. (1972) Sporulation and enterotoxin production by mutants of Clostridium perfringens. J. Bacteriol 110, 378^391. Finlay, W. J. J., Logan N. A. and Sutherland A. D. (1999) Semi-automated metabolic staining assay for Bacillus cereus emetic toxin. Appl. Environ. Microbiol. 65, 1811^1812. Finlay, W. J. J., Logan N. A. and Sutherland A. D. (2000) Bacillus cereus produces most emetic toxin at lower temperatures. Lett. Appl. Microbiol. 31, 385^389. Glatz, B. A. and Goepfert, J. M. (1976) De¢ned conditions for synthesis of Bacillus cereus enterotoxin by fermenter-grown cultures. Appl. Environ. Microbiol. 32, 400^404. Goldberg, J. J., Bramley, A. J., Sjogren, R. E. and Pankey, J.W. (1994) E¡ects of temperature and oxygen tension on growth of Escherichia coli in milk. J. Dairy Sci. 77, 3338^3346. Granum, P. E. and Lund,T. (1997) Bacillus cereus and its food poisoning toxins. FEMS Microbiol. Lett. 157, 223^228.
Hughes, S., Bartholomew, B., Hardy, J. C. and Kramer, J. M. (1989) Potential application of a Hep-2 cell assay in the investigation of Bacillus cereus emetic-syndrome food poisoning. FEMS Microbiol. Lett. 52, 7^12. Johnson, K. M., Nelson, C. L. and Busta, F. F. (1982) Germination and heat resistance of Bacillus cereus spores from strains associated with diarrhoeal and emetic food-borne illnesses. J. Food Sci. 47, 1268^1271. Labbe, R. G. and Duncan, C. L. (1977) Spore coat protein and enterotoxin synthesis in Clostridium perfringens. J. Bacteriol 131, 713^715. Logan, N. A., Capel, B. J., Melling, J. and Berkeley, R. C.W. (1979) Distinction between emetic and other strains of Bacillus cereus using the API system and numerical methods. FEMS Microbiol. Lett. 5, 373^375. Mahler, H., Pasi, A., Kramer, J. A., Schulte, P., Scoging, A. C., Bar,W. and Krahenbuhl, S. (1997) Fulminant liver failure in association with the emetic toxin of Bacillus cereus. N. Engl. J. Med. 336, 1142^1148. Mikkola, R., Saris, N. E. L., Grigoriev, P. A., Andersson, M. A. and SalkinojaSalonen, M. S. (1999) Ionophoretic properties and mitochondrial e¡ects of cereulideFThe emetic toxin of Bacillus cereus. Eur. J. Biochem. 263, 112^117. Nichols, G. L., Little, C. L., Mithani, V. and de Louvois, J. (1999) The microbiological quality of cooked rice from restaurants and take-away premises in the United Kingdom. J. Food Prot. 62, 877^882. Ryu, S. and Labbe, R. G. (1989) Coat and enterotoxinrelated proteins in Clostridium perfringens spores. J. Gen. Microbiol. 135, 3109^3118. Shinagawa, K., Otake, S., Matsusaka, N. and Sugii, S. (1992) Production of the vacuolation factor of Bacillus cereus isolated from vomiting-type food poisoning. J.Vet. Med. Sci. 54, 443^446. Spira,W. M. and Silverman, G. J. (1979) E¡ects of glucose, pH, and dissolved oxygen tension on Bacillus cereus growth and permeability factor production in batch culture. Appl. Environ. Microbiol. 37, 109^116. Szabo, R. A., Spiers, J. I. and Akhtar, M. (1991) Cell culture detection and conditions for production of a Bacillus cereus heat-stable toxin. J. Food Prot. 54, 272^276. Te Gi¡el, M. C., Beumer, R. R., Granum, P. E. and Rombouts, F. M. (1997) Isolation and characterisation of Bacillus cereus from pasteurised milk in household refrigerators in the Netherlands. Int. J. Food Microbiol. 34, 307-318. Yokoyama, K., Ito, M., Agata, N., Isobe, M., Shibayama, K., Horii, T. and Ohta, M. (1999) Pathological e¡ect of cereulide, an emetic toxin of Bacillus cereus, is reversible in mice. FEMS Immunol. Med. Microbiol. 24, 115^120.
doi:10.1006/yfmic.504
ORIGINAL ARTICLE
Bacillus cereus emetic toxin production in relation to dissolved oxygen tension and sporulation W. J. J. Finlay1;2, N. A. Logan1 and A. D. Sutherland1;
Seven emetic toxin-producing strains of Bacillus cereus were examined for growth, sporulation and toxin production in skim milk medium in shaken and unshaken batch cultures under aerobic, microaerobic and anaerobic atmospheres. High levels of toxin production were detected in aerobic and microaerobic cultures. Static cultures yielded 90% less toxin than equivalent shaken cultures. Emetic toxin production was detected in aerobic and microaerobic cultures when bacterial counts reached 4 6?0 log10 cfu ml 1, which also coincided with spore production. However, no correlation was found between spore count and toxin production (R2 =0?032). In anaerobic culture, production of emetic toxin was undetectable and the dissolved oxygen tension in the growth medium fell below 125746 ppm within 6 h of inoculation. In aerobic culture, most toxin was produced between 16 and 22 h of incubation, when the dissolved oxygen tension had decreased to 60 70 ppm.Therefore, while O2-dependent respiration is fundamentally required for production of the emetic toxin, most toxin production occurs when little free oxygen is available. # 2002 Elsevier Science Ltd. All rights reserved.
Introduction Bacillus cereus is capable of producing several toxins, including a necrotizing enterotoxin, an emetic toxin, phospholipases, proteases and haemolysins. Of the food-poisoning toxins, the emetic toxin 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). Fulminant liver failure has also been shown to be inducible in laboratory mice using synthetically produced emetic toxin (Yokoyama et al. 1999). The emetic toxin is an extremely stable * Corresponding author. Fax: +44 141 331 3208; E-mail: [email protected] 0740 -0020/02/050423+08 $35.00/0
dodecadepsipeptide, which acts as a potassium ionophore (Mikkola et al. 1999), and which is resistant to proteolytic degradation, pH extremes and high temperature, surviving 1211C for 90 min (Granum and Lund 1997). In western countries, B. cereus emetic food poisoning is associated mainly with Chinese fried rice dishes and farinaceous foods such as pasta and noodles. One recent study showed that 2% of pre cooked rice from take-away restaurants in the UK contained B. cereus at 4104 cfu g 1 (Nichols et al. 1999). B. cereus is also associated with dairy produce; Te Gi¡el et al. (1997) showed that 40% of the skim milk samples they collected in the Netherlands contained mesophilic B. cereus at levels between 50 and 5000 cfu ml 1. r 2002 Elsevier Science Ltd. All rights reserved.
Received: 9 April 2002 1 School of Biological and Biomedical Sciences, Glasgow Caledonian University, Cowcaddens Road, Glasgow G4 OBA, Scotland, UK 2 Current address: Centre for Biologics Evaluation and Research, US Food and Drug Administration, 29 Lincoln Drive, Bethesda, Maryland 20892, USA
424 W. J. J. Finlay et al.
The minimum amino acid nutritional requirements for emetic toxin production have been described by Agata et al. (1999), and we have recently reported the ability of B. cereus strains to produce large amounts of emetic toxin in skim milk medium (SMM) under lowered growth temperatures (Finlay et al. 2000). However, other fundamental environmental requirements for production of the emetic toxin have not yet been investigated fully. Several researchers (Hughes et al. 1989, Szabo et al. 1991, Agata et al. 1996) have shown that high levels of emetic toxin production in liquid cultures require vigorous shaking (up to a maximum of 200 rpm). This suggested that oxygenation of the culture medium in vitro may be an important stimulus for emetic toxin production. If this is the case, then oxygen tension in the microenvironment may also be of critical importance in production of the emetic toxin in foods. Enterotoxin production is known to be associated with the formation of spores in other genera such as Clostridium (Duncan et al. 1972, Labbe and Duncan 1977, Ryu and Labbe 1989). Andersson et al. (1998) also found that growth of emetic toxin-producing B. cereus strains on tryptic soy agar plates for 10 days at 281C created mostly sporulated and lysed cells and yielded 0?5 ng of puri¢ed emetic toxin per 104 cfu of B. cereus. They also showed that cooked rice samples, in which emetic toxin had been produced, contained mainly sporulated and lysed cells with very few vegetative cells. These ¢ndings suggested that production of the B. cereus emetic toxin might also be associated with sporulation. To investigate these factors, the growth, sporulation, oxygen consumption and toxin production of seven known emetic B. cereus strains were examined in static and shaken batch cultures under aerobic, microaerobic and anaerobic conditions.
Materials and Methods Bacterial strains The seven strains studied have previously been shown to produce emetic toxin (Finlay et al. 1999, 2000). Six of the strains (F4810/72, F3748/
75, F3744/75, F4562/75, F4552/75, and F2427/76) have also been 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 obtained originally from the Central Public Health Laboratory, Colindale, London, UK and have been maintained in the Logan Bacillus collection, now at Glasgow Caledonian University, since 1978.
Culture conditions Single colonies of each strain tested were selected from streak plates on Nutrient Agar (NA) (Oxoid, UK) (301C/overnight) and inoculated into 3 ml Nutrient Broth (NB) (Oxoid, UK). These cultures were then incubated overnight at 301C with 200 rpm orbital shaking. Cultures were diluted in NB to give ¢nal concentrations of approximately 1 103 cfu ml 1 in triplicate 50 ml volumes of SMM (Oxoid, UK) in 500 ml conical £asks. Skim milk medium has been shown to be the optimum medium for emetic toxin production (Szabo et al. 1991, Finlay et al. 1999). These cultures were placed immediately in aerobic (ambient O2 ), microaerophilic or anaerobic environments. Modi¢ed oxygen conditions were created in Gas Pak jars with a microaerobic catalyst sachet or anaerobic catalyst sachet to produce 410% O2 and 0% O2, respectively (all BBL Microbiology Systems, UK), with the exception that static anaerobic cultures were incubated under 0% O2 in an anaerobic cabinet (Don Whitley Ltd, UK). Anaerobic conditions were con¢rmed using indicator strips (Oxoid, UK). Triplicate cultures of each strain under aerobic, microaerobic and anaerobic conditions were incubated at 301C for 24 h with or without shaking (200 rpm).
B. cereus emetic toxin 425
Continuous measurement of oxygen tension in stirred batch cultures Probes for the measurement of dissolved oxygen tension (DOT) are ine¡ective in static liquids owing to the depletion of O2 in the immediate vicinity of the probe by the electrode itself. As measurements of DOT in static cultures would therefore be misleading, DOT was only measured in agitated (stirred) cultures; it was measured in parts per million (ppm). Cultures of strain F4810/72 were prepared as described above but with the addition of a sterile 30 mm rod-shaped magnetic stirrer. Vigorous magnetic stirring was applied to the culture to encourage oxygenation of the medium and so mimic the rapid shaking conditions described by other authors (Hughes et al. 1989, Szabo et al. 1991, Agata et al. 1996).This allowed the e¡ective use of a ‘Micro O2 probe’ (Lazar Research Laboratories Inc., USA), to monitor DOT in the culture continuously. A Gas Pak (BBL) jar was modi¢ed to allow DOT measurement; a small hole, su⁄cient to accommodate the electrical wire extending from the O2 probe, was drilled in the lid of the jar. This hole was then sealed on both sides using epoxy resin; to stabilize the O2 probe and to ensure an airtight seal. An uninoculated negative control culture (to measure normal DOT levels in SMM at 301C over the incubation period) was created using SMM with the addition of 20 mg ml 1 Gentamycin (Sigma, UK), to inhibit the growth of any bacterial contaminants. Digital DOT readings were taken regularly, and DOT was also recorded continually on a chart recorder (Linseis, UK), for 24 h with constant stirring under aerobic, microaerobic or anaerobic conditions. Experiments using the di¡erent growth conditions were performed on three separate occasions. All cultures from DOT experiments were analysed for bacterial growth and toxin production as described below.
Total counts, spore counts and toxicity assay Two 1 ml samples were taken aseptically from each batch culture after 24 h of incubation. One sample was placed in a water bath at 801C for 10 min in order to kill vegetative cells of B.
cereus and provide a presumptive count for spores (Johnson et al. 1982).The other 1 ml sample was unheated, and used to provide a viable count. Log serial dilutions were prepared in NB and viable or spore counts were estimated by plating duplicate 20 ml drops of each dilution on NA. The remainder of the culture was then centrifuged (4500 g 40 min 1 41C 1 ) to remove bacteria and large particulates. Supernatants were decanted and autoclaved (1211C for 15 min) to sterilize and to denature heat-labile toxins such as B. cereus diarrhoeal toxin. Supernatants were then tested for the emetic toxin using the MTT metabolic staining assay described previously (Finlay et al. 1999).
Growth curve of aerobic culture A growth curve for shaken aerobic culture was also performed using triplicate cultures of strain F4810/72, prepared as above. One-millilitre samples were removed aseptically every 2 h, up to 24 h of incubation. Two 100 ml aliquots were removed from this sample and used as described above to provide spore and viable counts. The remaining 800 ml of sample was then used to prepare supernatant for toxicity testing, as described above.
Statistical analysis The results of viable counts and spore counts (derived from triplicate cultures of all of the bacterial strains) were expressed as log10 mean7standard deviation. Toxin titres of the individual strains were expressed as means of their reciprocal toxin titres, as described previously (Finlay et al. 1999). Grand mean reciprocal toxin titre7grand mean standard deviation values (to show overall variability) were also calculated for triplicate cultures of all strains. A paired two-tailed t-test was used to evaluate the statistical signi¢cance of changes in growth, sporulation or toxicity values with change in atmospheric conditions. Analysis of the relationship between spore counts and emetic toxin production across the range of atmospheric conditions was performed by regression analysis of paired values
426 W. J. J. Finlay et al.
(95% con¢dence interval) for all bacterial strains.
Results The results from triplicate cultures of each of the seven strains, when tested under the same conditions, were very similar and data are, therefore, presented as mean values. Shaken aerobic and microaerobic conditions gave similar mean viable counts for all strains at 24 h (8?670?3 and 8?570?2 log10 cfu ml 1 respectively) (Table 1). Static aerobic and microaerobic growth also yielded similar mean viable counts at 24 h (8?270?5 and 7?870?8 log10 cfu ml 1 , respectively) (Table 1). However, all strains grew less abundantly under shaken and static anaerobic conditions, producing mean viable counts which were signi¢cantly (Po0?01) lower than equivalent cultures in aerobic or microaerobic conditions (7?470?2 and 7?370?2 log10 cfu ml 1 , respectively) (Table 1). No signi¢cant rise in total count was observed under any of the atmospheric conditions when the cultures were shaken (P40?05) (Table 1). Toxin was detectable in shaken and static aerobic and microaerobic cultures but not in static or shaken anaerobic cultures. This was despite mean viable counts in anaerobic culture being consistently 46?0 log10 cfu ml 1 , which is su⁄cient to produce detectable toxin in shaken aerobic culture (Hughes et al. 1989, Agata et al. 1996, Finlay et al. 2000). Mean spore counts for all strains under all conditions were typically 3 orders of magnitude lower than their respective mean viable
counts (Table 1). Shaken anaerobic conditions produced spore counts similar to those observed in shaken aerobic growth (4?870?9 and 4?870?5 log10 cfu ml 1 , respectively). Shaken microaerobic growth however, gave signi¢cantly (Po0?01) increased mean spore counts (6?770?3 log10 cfu ml 1 ) (Table 1), without leading to signi¢cantly (P40?05) higher toxicity than that produced in shaken aerobic growth (Table 1). Static aerobic and microaerobic conditions gave respective spore counts of 5?170?1 and 5?270?4, which were not signi¢cantly (P40?05) di¡erent from those observed in shaken aerobic culture (Table 1). Despite this, toxin levels dropped by 89% and 87%, respectively, in static aerobic and microaerobic cultures, when compared to shaken cultures (Table 1). Spore counts in static anaerobic culture were not signi¢cantly di¡erent (P40?05) from those observed in static aerobic and microaerobic culture (Table 1). Regression analysis of spore counts and emetic toxin production across the range of atmospheric conditions, therefore, found no correlation between sporulation level and toxin production (R2 = 0?032). However, when a growth-curve analysis for strain F4810/72 was performed, it showed that emetic toxin production in aerobic culture became detectable after 6 h of incubation, which coincided with spores becoming evident (mean spore count of 2?470?4 log10 cfu ml 1 ) (Fig. 1). All strains produced high levels of toxin under both aerobic and microaerobic conditions (Table 1). Mean toxin titres from shaken microaerobic cultures were 20% lower than those obtained from shaken aerobic cultures. In comparison with static cultures, shaken cultures
Table 1. Mean log10 viable count, spore count and reciprocal toxin titre for seven strains of Bacillus cereus under varying atmospheric conditions Culture conditions Aerobic shaken Aerobic static Microaerobic shaken Microaerobic static Anaerobic shaken Anaerobic static
Total log10 cfu ml 8?6 (70.3)* 8?2 (70.5) 8?5 (70?2) 7?8 (70?8) 7?4 (70?2) 7?3 (70?2)
1
Spore log10 cfu ml 4?8 (70.5) 5?3 (70?1) 6?7 (70?3) 5?2 (70?4) 4?8 (70?4) 4?8 (70?9)
1
Reciprocal toxin titre 3191 (710) 358 (7138) 2547 (7781) 342 (7102) 0 0
*Error values in parentheses are standard deviation for triplicate cultures of all the seven strains analysed (i.e. n = 21).
B. cereus emetic toxin 427
viable count
spore count
toxin titre
log10 count ml-1
8
2000
7 6
1500
5
1000
4 500
3 2
Reciprocal toxin titre
2500
9
0 2
4
6
8 10 12 14 16 18 20 22 24 Time (hours)
Figure 1. Mean (7s.e.) viable count, spore count and reciprocal toxin titres for B. cereus strain F4810/72 at 301C over 24 h, under aerobic growth conditions.
gave signi¢cant (Po0?01) increases in toxicity under both microaerobic and aerobic conditions (Table 1). Microaerobic cultures showed less consistency in toxin titres following shaken incubation (Table 1). None of the strains produced toxin under either static or shaken anaerobic conditions (Table 1). The modi¢ed Gas Pak jar remained airtight when anaerobic atmospheric conditions were induced, as evidenced by a DOT of o20 ppm and by the positive reaction of anaerobic indicator strips. Also, mean viable counts, spore counts and toxicity values were not signi¢cantly di¡erent (P40?05) from those observed in unmodi¢ed gas jars. All cultures were found to have initial DOT values of 762724 ppm at 301C. This is in agreement with the normal DOT in distilled water of 760 ppm at 301C and at atmospheric pressure of 750 mm Hg. In the negative control stirred aerobic culture, DOT was maintained at a constant level over 24 h, with an initial value of 759714 ppm and a ¢nal value of 761717 ppm.Therefore, the observed reduction in DOT values below 760 ppm in aerobic cultures (Fig. 2) was considered to re£ect oxygen consumption by bacterial growth. In aerobic culture, DOT dropped slowly to 710712 ppm during the ¢rst 4 h of incubation and then decreased more rapidly to 2767 21ppm after 8 h of incubation reaching its minimum value of 68711ppm at 22 h. In microaerobic culture, the catalyst sachet brought about a rapid reduction of DOT to 461727 ppm after 2 h, and a slower reduction to 183745 ppm at
8 h; DOT then continued to drop steadily, reaching 6479 ppm after 16 h of incubation and 57713 ppm after 24 h of incubation. In anaerobic cultures, the catalyst sachet brought about a rapid reduction of DOT to 348742 ppm in 2 h. Reduction in DOT then slowed, reached 2578 ppm after 8 h, and maintained this level until 24 h of culture, when DOT was 16711ppm. Aerobic growth-curve data (Fig. 1) showed that toxin production became detectable (reciprocal toxin titer 574) after 6 h of incubation, when the viable count had reached 6?170?1. At this time, DOT values for the aerobic, microaerobic and anaerobic cultures were 561713, 278741 and 125746 ppm, respectively (Fig. 2). In aerobic growth, levels of emetic toxin rose to a mean reciprocal toxin titre of 6827295 after 18 h of incubation, by which point cultures had entered stationary phase (Fig. 1) and DOT had declined to 72726 (Fig. 3). Toxin production in aerobic culture then rose considerably, with no observed increase in DOT, reaching a maximum level of 204870 after 22 h of incubation (Fig. 3).
Discussion Labbe and Duncan (1977) demonstrated that enterotoxin production in Clostridium perfringens occurs coincidentally with sporulation. Shinagawa et al. (1992) have also suggested that B. cereus emetic toxin is produced only when sporulation is underway, being detected 5 h after sporulation was ¢rst observed. In the present study, growth-curve analysis of strain F4810/72 in SMM also revealed that emetic toxin became detectable as spore production began. However, the spore counts evident when emetic toxin production became detectable (2?470?4 log10 cfu ml 1 ) were more than 2?0 log10 lower than those associated (5?0 log10 cfu ml 1 ) with clostridial enterotoxin production (Labbe and Duncan 1977), and more than 4 log lower than the ¢gure of 6?7 log10 cfu ml 1 reported to be required for B. cereus emetic toxin production in rice broth culture (Shinagawa et al. 1992). Spore counts observed in this study were generally more than 3 log10 lower than the viable count levels.
428 W. J. J. Finlay et al.
Dissolved Oxygen Tension (log10 ppm)
8 7 6 5 4 3 2 1 0 2
4
6
8
10
12
14
16
18
20
22
24
TIME (Hours)
Figure 2. DOT measurement aerobic (
), microaerobic ( ) and anaerobic growth conditions, for B. cereus strain F4810/72, in stirred culture at 301C over 24 h. 8
2250
8.5
2000
8.0
6
1750
7.5
5
1500
(
)
1000 3 750 2
7.0 6.5 6.0 5.5
log10 viable count
1250 4
Reciprocal Toxin Titre
Dissolved Oxygen Tension (log10 ppm)
7
5 500 4.5
1 250 0
4.0
0 2
4
6
8
10
12
14
16
18
20
22
24
TIME (Hours)
Figure 3. DOT measurements under aerobic growth conditions ( ), versus mean growth (B) and emetic toxin production (&) under aerobic conditions, for B. cereus strain F4810/72, in stirred culture at 301C over 24 h.
In shaken microaerobic growth, sporulation occurred at a higher level than in aerobic growth, without a corresponding increase in toxin production in relation to aerobic culture. Static aerobic or microaerobic culture also produced spore levels comparable to those
achieved in shaken aerobic growth, but toxin levels were almost 90% lower than those produced by shaken culture. Also, both shaken and static anaerobic cultures yielded similar numbers of spores to those achieved by shaken aerobic cultures. Regression analysis thus
B. cereus emetic toxin 429
showed no correlation (R2 = 0?032) between levels of sporulation and emetic toxin production in the di¡erent cultures. As no toxin was detected in any anaerobic culture, these data support our earlier ¢ndings that B. cereus spore formation can proceed without emetic toxin production (Finlay et al. 2000). It also supports our suggestion that the emetic toxin, while becoming detectable coincidentally with spore development, is not produced in direct association with the sporulation process. Production of B. cereus enterotoxin has been shown to proceed under anaerobic growth conditions (Glatz and Goepfert 1976), but the present study shows that the emetic toxin is not produced under anaerobic conditions. These ¢ndings, therefore, suggest that production of the emetic toxin is unlikely to be regulated by the same mechanism as the B. cereus enterotoxins. Analysis of the growth curve of strain F4810/ 72 under aerobic culture conditions showed that emetic toxin production only became detectable when viable counts reached 6?0 log10 cfu ml 1, at 6 h of incubation. This is consistent with the ¢ndings of Hughes et al. (1989) and Agata et al. (1996), who showed that emetic toxin production begins in SMM after 6^8 h shaken incubation at 301C, at a viable count of roughly 6?0 log10 cfu ml 1. It has also recently been reported that toxin production occurs at 151C in SMM, when viable count reaches 6?0 log10 cfu ml 1 or above (Finlay et al. 2000). These ¢ndings suggest that expression of the emetic toxin is either regulated by cell density, or that toxin concentration only reaches a detectable level when counts above 6?0 log10 cfu ml 1 are achieved. Glatz and Goepfert (1976) have shown that, in the growth of B. cereus in 300 ml cultures performed in fermenter vessels (with agitation), DOT drops rapidly during exponential phase growth, and conditions become anaerobic once cultures enter stationary phase. As B. cereus enters late exponential phase O2 demand increases, rendering maintenance of a high DOT level di⁄cult (Spira and Silverman 1979), even with rapid agitation and sparging of the medium with O2 (Bauer and Ziv 1976). The DOT curve observed in the present study, for growth in agitated aerobic culture, shows that rapid O2 consumption also occurs in small-
scale batch (50 ml) culture and that in late exponential and stationary phase growth, low levels of DOT occur (72726 ppm). Rates of O2 consumption in milk for B. cereus in the present study were similar to those observed for Escherichia coli by Goldberg et al. (1994); in aerobic and microaerobic fermenter cultures, rapid reduction of DOT occurred during exponential phase growth and the culture became anaerobic once the population of E. coli achieved a viable count of 6?0 log10 cfu ml 1 . Measurement of DOT in stirred cultures revealed that toxin production could be detected after 6 h of incubation, when oxygen was still freely available in both aerobic and microaerobic cultures (561713 and 278741ppm, respectively), while in anaerobic culture DOT had dropped to 125746 ppm by this time. However, the majority of toxin was produced between 16 and 22 h of incubation, when aerobic and microaerobic cultures had entered stationary phase and DOT was only 60^70 ppm. Culture in the presence of an anaerobic catalyst sachet reduced DOT to 25 ppm within 8 h, meaning that little or no free oxygen remained in the medium, precluding production of the toxin as the organisms had entered a state of anaerobic respiration. The low DOT that was observed in the late log/stationary phases of growth in cultures performed under aerobic and microaerobic conditions (Fig. 3) was almost certainly caused by bacterial oxygen consumption outstripping the oxygen input achieved by agitation. The amount of oxygen being provided by agitation was, however, su⁄cient to allow aerobic respiration of the cultures (and subsequent emetic toxin production), as even at sub-microaerobic DOT, the di¡usion of O2 into the cytoplasm of bacteria occurs at a rate su⁄cient to allow catabolism (Arras et al. 1998). The ¢ndings presented in this study show that static microaerobic cultures produce comparable amounts of emetic toxin to static aerobic cultures, and that shaken microaerobic and aerobic cultures also produce very similar toxin levels. Free oxygen levels are very low in late log phase and stationary phase shaken cultures, even with rapid agitation of the culture medium, and toxin production in static cultures is 90% lower than that observed in shaken cultures. This, therefore, suggests that
430 W. J. J. Finlay et al.
shaking maintains the minimal levels of DOT required to produce the emetic toxin. Static culture does not allow e⁄cient O2 distribution in the growth medium and this leads to a lower level of toxin production. The ¢ndings reported here imply that while O2 -dependent respiration is required fundamentally for production of the emetic toxin, most toxin production occurs when little free oxygen is available in the growth medium.
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Hughes, S., Bartholomew, B., Hardy, J. C. and Kramer, J. M. (1989) Potential application of a Hep-2 cell assay in the investigation of Bacillus cereus emetic-syndrome food poisoning. FEMS Microbiol. Lett. 52, 7^12. Johnson, K. M., Nelson, C. L. and Busta, F. F. (1982) Germination and heat resistance of Bacillus cereus spores from strains associated with diarrhoeal and emetic food-borne illnesses. J. Food Sci. 47, 1268^1271. Labbe, R. G. and Duncan, C. L. (1977) Spore coat protein and enterotoxin synthesis in Clostridium perfringens. J. Bacteriol 131, 713^715. Logan, N. A., Capel, B. J., Melling, J. and Berkeley, R. C.W. (1979) Distinction between emetic and other strains of Bacillus cereus using the API system and numerical methods. FEMS Microbiol. Lett. 5, 373^375. Mahler, H., Pasi, A., Kramer, J. A., Schulte, P., Scoging, A. C., Bar,W. and Krahenbuhl, S. (1997) Fulminant liver failure in association with the emetic toxin of Bacillus cereus. N. Engl. J. Med. 336, 1142^1148. Mikkola, R., Saris, N. E. L., Grigoriev, P. A., Andersson, M. A. and SalkinojaSalonen, M. S. (1999) Ionophoretic properties and mitochondrial e¡ects of cereulideFThe emetic toxin of Bacillus cereus. Eur. J. Biochem. 263, 112^117. Nichols, G. L., Little, C. L., Mithani, V. and de Louvois, J. (1999) The microbiological quality of cooked rice from restaurants and take-away premises in the United Kingdom. J. Food Prot. 62, 877^882. Ryu, S. and Labbe, R. G. (1989) Coat and enterotoxinrelated proteins in Clostridium perfringens spores. J. Gen. Microbiol. 135, 3109^3118. Shinagawa, K., Otake, S., Matsusaka, N. and Sugii, S. (1992) Production of the vacuolation factor of Bacillus cereus isolated from vomiting-type food poisoning. J.Vet. Med. Sci. 54, 443^446. Spira,W. M. and Silverman, G. J. (1979) E¡ects of glucose, pH, and dissolved oxygen tension on Bacillus cereus growth and permeability factor production in batch culture. Appl. Environ. Microbiol. 37, 109^116. Szabo, R. A., Spiers, J. I. and Akhtar, M. (1991) Cell culture detection and conditions for production of a Bacillus cereus heat-stable toxin. J. Food Prot. 54, 272^276. Te Gi¡el, M. C., Beumer, R. R., Granum, P. E. and Rombouts, F. M. (1997) Isolation and characterisation of Bacillus cereus from pasteurised milk in household refrigerators in the Netherlands. Int. J. Food Microbiol. 34, 307-318. Yokoyama, K., Ito, M., Agata, N., Isobe, M., Shibayama, K., Horii, T. and Ohta, M. (1999) Pathological e¡ect of cereulide, an emetic toxin of Bacillus cereus, is reversible in mice. FEMS Immunol. Med. Microbiol. 24, 115^120.