Prevalence and Levels of Bacillus cereus Emetic Toxin in Rice Dishes Randomly Collected from Restaurants and Comparison with the Levels Measured in a Recent Foodborne Outbreak

Abstract

Whereas the prevalence of Bacillus cereus emetic strains in the environment has been shown to be very low, there is a lack of information on the prevalence of its toxin, cereulide, in food. Yet, the rice leftovers of a family outbreak which occurred after the consumption of dishes taken away from an Asian restaurant revealed significant amounts of cereulide, reaching up to 13,200 ng/g of food. The occurrence of cereulide in rice dishes collected from various restaurants was therefore evaluated using the liquid chromatography coupled with tandem mass spectrometry method, which allows for the direct quantification of the toxin in food. The cereulide prevalence was found to be 7.4% when samples were analyzed at the day of sampling, but reached 12.9% when exposed to temperature abuse conditions (25°C). The cereulide concentrations observed in cooked rice dishes were low (approximately 4 ng/g of food). However, since little is known yet about the potential chronic toxicity of cereulide, one needs to be very careful and vigilant.

Introduction

B acillus cereus is a spore-forming pathogen that possesses a facultative anaerobic fitness and the ability to grow in a large range of temperature (from 5°C to 45°C). This explains its ubiquitous presence in environments and in food products. B. cereus can cause two types of foodborne illnesses: emesis, caused by cereulide, is mainly associated with starchy foods (Schoeni and Wong, 2005), and diarrheal syndromes are induced by a variety of enterotoxins and are associated with protein-rich foods. Emetic strains of B. cereus have been shown to be very rare in the environment (Altayar and Sutherland, 2006), and several studies have determined this low frequency to be approximately 1–2% (Hoton et al., 2009; Nguyen-The, 2009). Yet, the incidence of B. cereus toxin–related food poisonings is steadily increasing in Europe (EFSA, 2009), and lethal cases of emetic intoxications, associated with the consumption of rice or pasta dishes, have been described worldwide (Dierick et al., 2005; Mahler et al., 1997; Naranjo et al., 2011; Shiota et al., 2010).

Detection of pathogenic organisms in food is a standard measure in industry and is the first control to be made after intoxication cases are signalled. Besides the detection of cereulide by biological or molecular assays, its quantification has so far not been frequently performed due to lack of accurate and sensitive methods. Recently though, a chemical method based on liquid chromatography coupled with tandem mass spectrometry (LC-MS²) has been developed for the appropriate and accurate quantification of cereulide in food matrices (Delbrassinne et al., 2011a; Bauer et al., 2010).

Cooked rice and its improper storage represent one of the major risk factors in emetic food poisoning. Indeed, rice is implicated in 95% of emetic food poisoning cases (Altayar and Sutherland, 2006; Ehling-Schulz et al., 2004; Schoeni and Wong, 2005; Shinagawa, 1990). Furthermore, two very recent prevalence studies (Chang et al., 2011; Perera and Ranasinghe, 2012) performed in Chinese-style restaurants revealed that B. cereus was frequently present (37.5% and 56%, respectively) in the served fried rice dishes. Unfortunately, the toxigenic potential of the isolated strains was not assessed. Since rice is regularly served as a main or side dish in many restaurants (among which Asiatic restaurants are quite representative), it is relevant to evaluate the incidence of cereulide in served rice dishes.

The aim of the present study was to assess the prevalence and concentration of cereulide in rice dishes collected in restaurants in several cities of Wallonia and Brussels. This quantification was performed thanks to an analytical method (LC-MS²) specifically targeting cereulide in food. Food samples recovered from a recent family outbreak due to emetic B. cereus were similarly analyzed to allow for comparison.

Methods

Food samples from family outbreak and from randomly picked restaurants

Two types of food samples were analyzed following a family outbreak: four samples from leftovers (white rice #1, fried rice, noodle sauce, and Wong Tong soup) that had been partially eaten, and three samples from inspection (white rice #2, noodles, and chickpeas) that were collected by food inspectors in the incriminated restaurant 24 h after the official complaint. The intoxication affected several members of the same family (ages 14–80) after ingestion of meals collected in an Asian take-away restaurant in Belgium. Eight of the 11 exposed people became ill within 2 h, with vomiting episodes, while some of them experienced additional diarrhea. One elderly person (80 years old) needed hospitalization.

In a small-scale survey study, a total of 54 randomly chosen restaurants were visited in Brussels and Wallonia during Spring and Summer 2011 (see supplementary Table S1 at www.liebertpub.com/fpd). Prepared rice dishes (13 white rice and 41 fried rice dishes) were collected and submitted to direct analysis (at the day of sampling or within 24 h of purchase). In order to evaluate the incidence of cereulide in rice in case of prolonged storage at temperature abuse, the food remnants of the analyzed restaurant samples were subsequently placed for 5 days in an incubator at 25°C.

Enumeration of B. cereus from food samples

The ISO7932 method was used for the B. cereus enumeration. After 24 h of incubation on Mannitol egg Yolk Polymyxin (MYP) agar media at 30°C, the typical pink colonies surrounded by a zone of lecithin hydrolysis were enumerated.

Extraction of cereulide from food samples and analysis by LC-MS²

The protocol recently described by Delbrassinne et al. (2011a) was applied, followed by injection in the LC-MS LCQ Deca-XP Plus ion trap mass analyser (ThermoFinnigan, USA) for cereulide determination. Valinomycin (Sigma-Aldrich, Belgium), diluted in methanol, was used as a surrogate standard for quantification. For each batch, the recovery efficiency was verified on the basis of valinomycin spiking (cooked rice spiked with valinomycin at 20 ng/g of rice), and a negative control (not contaminated cooked rice) was also included for evaluating the matrix interferences. The presence of cereulide was evaluated by monitoring the following m/z values: 1125.3, 1153.3, and 1170.5 in the MS² mode of analysis. The chromatograms were smoothed thanks to a Gaussian function.

Results

Analysis of samples implicated in a Belgian family outbreak

The food samples related to a recent family outbreak in Belgium were analyzed for their bacterial content and cereulide concentrations. B. cereus was present among six (fried rice, Wong Tong soup, noodle sauce, chickpeas, and two white rice dishes) of the seven collected samples and was absent in the noodles, as indicated in Table 1. The highest counts (2.4×10⁷; 1.5×10⁷; 1.3×10⁷, and 2.8×10⁵ CFU/g) were found in the leftovers eaten by the family (white rice dish #1, fried rice dish, soup, and noodle sauce, respectively). In the other food products that were collected in the same restaurant 24 h after the complaint (inspection samples), the counts were at least 10³ to 10⁴ times lower than in the leftovers.

Table 1.

Bacillus cereus Enumeration and Cereulide Determination in Samples Related to the Recent Belgian Food Poisoning

	Samples	B. cereus counts (CFU/g)	Cereulide concentration (ng eq/g)
Leftovers^a	White rice #1	2.4E+07	8,693
	Fried rice	1.5E+07	13,184
	Soybean soup	1.3E+07	16
	Noodle sauce	2.8E+05	152
Inspection samples^b	White rice #2	3.6E+04	9
	Chickpea	3.0E+03	<LOD
	Noodles	<1.0E+02	ND

Leftovers were directly consumed by the family after delivery and then stored in the households fridge.

Inspection samples were collected by food inspectors from restaurant and stored in the lab fridge (4°C) until analysis. The detection limit (LOD) for LC-MS² method was 0.5 ng/g. ND, not determined.

B. cereus counts (enumerated on MYP medium) and cereulide production (assayed by LC-MS² and expressed in ng valinomycin equivalent/g of food) are presented.

The six samples containing B. cereus also contained cereulide with the exception of the chickpea sample. Cereulide was found in concentrations ranging from 9 to 13,184 ng/g (Table 1). As a general trend, the leftovers contained more cereulide than the inspection samples. The fried and white rice #1 displayed the highest cereulide concentration (13,184 and 8,693 ng/g, respectively). The obvious discrepancy between these concentrations measured in the rice leftovers and the concentrations found in the white rice #2 and noodle sauce (9 and 152 ng/g, respectively) are in accordance with the lower B. cereus counts. Interestingly, the soybean soup (one of the leftovers of the outbreak) and the chickpea sample (inspection sample) displayed B. cereus counts but contained only low or no amounts of cereulide.

Incidence of B. cereus–like organisms in restaurant rice samples

A small scale study was performed in 54 Belgian restaurants in order to evaluate B. cereus presence and the potential cereulide production in rice dishes. Presumptive B. cereus strains were found in 10 of the 54 samples (18.5%) when analyzed within 24 h after sampling. The concentrations of B. cereus were low and ranged from 100 to 3,500 CFU/g. After 5 days of incubation at 25°C, 41/54 rice samples (75.9%) contained B. cereus at concentrations ranging from 2×10⁵ to 2×10⁹ CFU/g.

Incidence of cereulide in restaurant rice samples

The rice dishes collected in the various restaurants were also analyzed by LC-MS² for the presence of cereulide, before and after 5 days of storage at 25°C. For all the analyses, the toxin recovery was always above 75%. As shown in Table 2, cereulide was detected in four samples (7.4%) prior to incubation and in three additional samples after incubation. The concentrations of cereulide were very low in all the samples: below 2 ng/g for the samples that were not incubated (samples 1–4) and below 4 ng/g for the samples stored at 25°C (samples 5–7). As indicated in Table 2, four samples that contained cereulide before incubation turned out to be negative after incubation at 25°C.

Table 2.

Cereulide Concentrations and Bacillus cereus Counts in the Seven Cereulide-Positive Rice Dishes Before and After Incubation at 25°C

Samples	1	2	3	4	5	6	7
Type of rice	Fried rice						White rice
Cereulide concentration (ng eq/g)^a	<LOQ	<LOQ	1.57	1.96	<LOD	<LOD	<LOD
Presumptive B. cereus (CFU/g)^a	<1.0E+02	3.0E+02	<1.0E+02	3.5E+03	<1.0E+02	<1.0E+02	<1.0E+02
Cereulide concentration (ng eq/g)^b	<LOD	<LOD	<LOD	<LOD	<LOQ	3.75	3.94
Presumptive B. cereus (CFU/g)^b	5.0E+07	6.4E+08	1.0E+07	2.7E+08	<1.0E+02	4.2E+08	4.8E+08

Cereulide was detected in seven dishes originating from seven distinct restaurants. Cereulide concentrations (assayed by LC-MS² and expressed in ng valinomycin equivalent/g of food) and B. cereus counts (by enumeration on MYP medium and expressed in cfu/g) are given before (^a) and after (^b) 25°C incubation. The limits of quantification (LOQ) and detection (LOD) for LC-MS² were 1 and 0.5 ng/g, respectively.

Discussion

The leftovers from the outbreak contained significant B. cereus counts but variable cereulide concentrations. Except for the rice dishes that displayed the highest toxin amount, low levels of cereulide were found in the soybean soup, and it could not be detected in the chickpea sample. That may be explained by a less efficient cereulide extraction, as suggested by additional spiking experiments performed with these food matrices (unpublished results). Another possible explanation is a lower cereulide production in these matrices. Food composition has indeed been pointed out to have an important influence on toxin production (Rajkovic et al., 2006).

Temperature abuse storage of the dishes in the restaurant may be at the origin of the outbreak since it was reported by the food inspectors that the prepared dishes were conserved in a badly refrigerated counter. The controlled temperature was around 9°C, whilst it has been reported that cereulide can be produced at temperatures as low as 8°C (Thorsen et al., 2009; Delbrassinne et al., 2011b). Furthermore, a B. cereus cross contamination may have occurred between dishes because of general bad hygiene: foods were uncovered in the refrigerated counter, dustbins were fully open, and the fridge, microwave, and many other surfaces were dirty, as reported by the food inspectors.

The cereulide amounts present in two rice dishes were comparable to those reported in one of the lethal cases (Naranjo et al., 2011) in which a spaghetti meal containing cereulide at a concentration of 14,800 ng/g caused the death of a 20-year-old man. The people involved in this family outbreak reported vomiting and some diarrea episodes, with hospitalization of the oldest person (80 years old). Cereulide is known to be highly toxic to certain groups such as children and elderly people. In this case, the children were not strongly affected because they had not completely eaten their meals. This underscored the importance of knowing the total amount of ingested toxin and the necessity to establish the toxic dose of cereulide for human beings (in μg/kg of body weight). Furthermore, the significant levels of cereulide measured in rice dishes from the outbreak justify the need for an accurate assessment of its prevalence in rice-based meals served in restaurants.

The low incidence of B. cereus (18.5%) found in the restaurant dishes at the time of consumption is not surprising and is in line with the findings of Kim et al. (2009), who could not detect any B. cereus contamination in rice without performing enrichment. After incubation, the same samples displayed a higher incidence of B. cereus (75.9%). However, samples that contained cereulide did not always display B. cereus counts. This might be explained by the elimination of vegetative cells in a reheating step of rice, which is a quite common practice in Asian restaurants. Part of the discrepancies between cereulide-positive samples before storage that were negative after storage (Table 2) could be due to the heterogeneity of B. cereus repartition and consequently of its cereulide production in the samples. Three samples were only detected positive after incubation which may be explained by the growth of emetic strains that were present in the samples. Cereulide production was probably promoted by suitable conditions. However, for sample 5, which contained small amounts of cereulide, no B. cereus could be detected because there was an overgrowth of other background bacteria which displayed yellow colonies (data not shown).

According to previous studies, summarized in Table 3, it is generally recognized that emetic strains of B. cereus are rather rare in the environment. However, these B. cereus isolates were obtained on specific media selecting presumptive strains. Emetic strains may be overlooked by those standard methods for B. cereus detection which are based on positive lecithinase and positive haemolysis. It is indeed now recognized that hemolysis is often weak or absent in the cereulide-producing B. cereus (Apetroaie et al., 2005; Kim et al., 2010; Pirhonen et al., 2005); hence, some lecithinase-negative emetic strains have also been found (Apetroaie et al., 2005). This implies that cultural methods, upon which the PCR and cytotoxic assays rely, are likely to lead to an underestimation of the emetic strains prevalence.

Table 3.

Review of Previous Studies on Bacillus cereus Emetic Strains Prevalence in the Environment and in Food

References	Method	Source	B. cereus isolates (n)	Emetic isolates (n)	Frequency of emeticB. cereus (%)
Ankolekar et al. (2009)	PCR	Raw rice from retail food store	83	0	0
Kim et al. (2009)	PCR	Cooked rice and sunsik (powdered raw grains)	163	0	0
De Jonghe et al. (2010)	PCR, cytotoxicity test on Vero cell line	Raw milk from 10 farms	7	0	0
Samapundo et al. (2011)	PCR	Lasagna, béchamel and bolognaise sauces, pasta, rice, minced beef and carrots	80	0	0
Hoton et al. (2009)	PCR, bioassay	Random food samples	1,863	28	1.5
N'guessan (2010)	PCR	Pasta, rice, meat, potato, milk, spices, and honey	409	6	1.5
Rahmati and Labbe (2008)	PCR	Seafood samples	62	1	1.6
Altayar and Sutherland (2006)	MTT assay on HEp-2 cells	Soil, animal faeces, raw and processed vegetables	177	3	1.7
Svensson et al. (2006)	RAPD-PCR, bioassay,	Farms and dairy plants	1,757	34	1.9
	LC-MS on strains^a		3,911	44	1.1
Rosenquist et al. (2005)	PCR	48901 RTE-foods	40	1	2.5
Shaheen et al. (2006)	BioassayLC-MS on strains^a	Dried infant formula	100	4	4
Messelhausser et al. (2010)	Real-time PCR	Ice creams in Bavaria	508 (samples)	24 (samples)	4.7
Wijnands et al. (2006)	HEP-2 cell test	Pastry, vegetables, RTE-foods, milk, meat and fish.	796	65	8.2

LC-MS analysis was performed on a selected number of strains from plates as a confirmation of the bioassay.

MTT, metabolic staining assay using the 3-(4,5,-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide as cell viability indicator; HEP-2, vacuolization assay using cells originating from human carcinoma of the larynx; bioassay, sperm motility assay using boar semen cells; RTE-foods, ready-to-eat foods.

Although some studies failed to isolate any emetic strains of B. cereus (Ankolekar et al., 2009; Kim et al., 2009; De Jonghe et al., 2010; Samapundo et al., 2011), the frequency of emetic strains was generally evaluated to be 1–2.5%, using PCR, bioassay, or cytotoxicity assays on both foodborne and environmental B. cereus strains (Rosenquist et al., 2005; Altayar and Sutherland, 2006; Svensson et al., 2006; Rahmati and Labbe, 2008; Hoton et al., 2009; N'guessan, 2010). However, as discussed above, the random selection of isolates or the choice of the hemolysis and/or lecithinase actitivies as a basis for the selection may also have led to an underestimation of the actual number of emetic strains. In addition, drawbacks can be associated with the use of PCR since a sample free of bacteria but containing the toxin (e.g., after heat treatment) might happen to yield a negative result. Similarly, the toxin genetic determinants present in the strains may be overlooked by specific primers because of variability in targeted DNA sequences, as observed by Hoton et al. (2009).

Interestingly, a few reports have indicated a slightly higher prevalence of cereulide-producing strains in specific food products (Table 3): approximately 4% of cereulide-producing strains were detected in dried infant formula (Shaheen et al., 2006) and German ice creams (Messelhausser et al., 2010), while the highest occurrence of emetic strains (8.2%) was found in Dutch retailed food samples (Wijnands et al., 2006), using HEp-2 cell assay. This frequency is five times higher than the generally reported 1.5% (Nguyen-The, 2009; Hoton et al., 2009). Unfortunately, cereulide concentrations in these samples were not determined, and no comparison could therefore be established with results of the present study. The levels of cereulide found in the randomly selected rice dishes were measured to be on the order of ng/g. However, the reported family outbreak should lead the authorities to emphasize the risk of cereulide food intoxication in cases where strict hygiene rules and cold storage temperatures were not appropriately followed.

Conclusion

To our knowledge, this is the first study on the prevalence of cereulide that directly targets the toxin in restaurant food; previous studies had mainly targeted cereulide-producing strains. Although it has been shown that emetic B. cereus strains are rare, cereulide was found in several rice dishes randomly collected from restaurants. These results suggest that the prevalence of cereulide is probably higher than previously thought. Although the cereulide concentrations found in these randomly picked dishes were low, cereulide may reach hazardous levels in rice, as indicated by the reported outbreak. Therefore, additional studies on factors that affect the growth of emetic B. cereus and the production of cereulide are needed. Due to the cereulide prevalence reported in the present study, it seems essential to follow strict temperature conditions for the storage of cooked rice in order to ensure food safety. This message should be more efficiently passed on to restaurants, catering, and domestic consumers.

Footnotes

Acknowledgments

We extend our gratefulness to Jacques Lhermitte for helpful technical assistance and to the Federal Agency for the Safety of the Food Chain for providing the food samples and for the inspection data following the visit of the restaurant. Thomas Vanzieleghem is also acknowledged for fruitful reviewing. Financial support by the Federal Public Service of the Belgian Science Policy is gratefully acknowledged.

Disclosure Statement

No competing financial interests exist.

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Supplementary Material

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Prevalence and Levels of Bacillus cereus Emetic Toxin in Rice Dishes Randomly Collected from Restaurants and Comparison with the Levels Measured in a Recent Foodborne Outbreak

Abstract

Introduction

Methods

Food samples from family outbreak and from randomly picked restaurants

Enumeration of B. cereus from food samples

Extraction of cereulide from food samples and analysis by LC-MS2

Results

Analysis of samples implicated in a Belgian family outbreak

Incidence of B. cereus–like organisms in restaurant rice samples

Incidence of cereulide in restaurant rice samples

Discussion

Conclusion

Footnotes

Acknowledgments

Disclosure Statement

References

Supplementary Material

Extraction of cereulide from food samples and analysis by LC-MS²