Potential of selected infant food formulas for production of Bacillus cereus emetic toxin, cereulide

Cereulide producing Bacillus cereus was isolated from randomly chosen commercial infant foods. The cereulide production in infant food formulas was investigated. When the reconstituted foods were inoculated with >10(5) cfu ml(-1) of cereulide producing B. cereus, 2 to 200 microg of cereulide per 100 ml of food accumulated during 24 h of non-refrigerated storage. The amount of cereulide measured in the foods by the accurate chemical assay (LC-MS) matched with that found by sperm micro assay, proving the cereulide was the sole heat stable toxin in the foods and present in its toxic form. The infant formulas containing both cereal and dairy ingredients were the most supportive for cereulide production. Cereulide accumulation was affected by the infant food composition as well as by the handling of the food. Diluting the reconstituted food with water resulted in increased toxin production expressed as mug per volume. More cereulide was accumulated when the food was incubated stationary compared with moderate shaking. The amount of cereulide accumulated within 24 h at room temperature per 100 ml of cereal and dairy or in rice-nondairy reconstituted infant formulas, inoculated with >or=10(5) cfu ml(-1) of B. cereus strain F4810/72, was higher or similar to the amounts reported for foods implicated in emetic type of food poisonings. Thus mishandling and temperature abuse of infant foods may cause food poisoning when emetic B. cereus is present.     

Emetic toxin-producing strains of Bacillus cereus show distinct characteristics within the Bacillus cereus group

One hundred representative strains of Bacillus cereus were selected from a total collection of 372 B. cereus strains using two typing methods (RAPD and FT-IR) to investigate if emetic toxin-producing hazardous B. cereus strains possess characteristic growth and heat resistance profiles. The strains were classified into three groups: emetic toxin (cereulide)-producing strains (n=17), strains connected to diarrheal foodborne outbreaks (n=40) and food-environment strains (n=43), these latter not producing the emetic toxin. Our study revealed a shift in growth limits towards higher temperatures for the emetic strains, regardless of their origin. None of the emetic toxin-producing strains were able to grow below 10 degrees Celsius. In contrast, 11% (9 food-environment strains) out of the 83 non-emetic toxin-producing strains were able to grow at 4 degrees Celsius and 49% at 7 degrees Celsius (28 diarrheal and 13 food-environment strains). non-emetic toxin-producing strains. All emetic toxin-producing strains were able to grow at 48 degrees Celsius, but only 39% (16 diarrheal and 16 food-environment strains) of the non-emetic toxin-producing strains grew at this temperature. Spores from the emetic toxin-producing strains showed, on average, a higher heat resistance at 90 degrees Celsius and a lower germination, particularly at 7 degrees Celsius, than spores from the other strains. No difference between the three groups in their growth kinetics at 24 degrees Celsius, 37 degrees Celsius, and pH 5.0, 7.0, and 8.0 was observed. Our survey shows that emetic toxin-producing strains of B. cereus have distinct characteristics, which could have important implication for the risk assessment of the emetic type of B. cereus caused food poisoning. For instance, emetic strains still represent a special risk in heat-processed foods or preheated foods that are kept warm (in restaurants and cafeterias), but should not pose a risk in refrigerated foods.     

Analytical methods for Bacillus cereus and other Bacillus species

Bacillus cereus can give rise to two distinct forms of foodborne disease, the emetic and the diarrhoeal syndromes. The emetic syndrome is believed to be associated with an emetic toxin pre-formed in food. Cooked rice is the most common vehicle, and the symptoms are similar to those of Staphylococcus aureus intoxication. The diarrhoeal type is caused by an enterotoxin and the symptoms generally parallel those of the Clostridium perfringens food poisoning. The heat resistance of B. cereus spores and the non-fastidious nature of the organism facilitates its survival and/or growth in a wide variety of foods. This review describes analytical methods available for the isolation, identification, and enumeration of the organism, in addition to details about biological and immunological methods for toxin assay. Data are also presented concerning the incidence and epidemiology of B. cereus food poisoning around the world, and especially in Japan.     

Genetic diversity, antimicrobial resistance, and toxigenic profiles of Bacillus cereus strains isolated from Sunsik

Bacillus cereus can cause emetic and diarrheal types of food poisoning, but little study has been done on the toxins and toxin-encoding genes of B. cereus strains isolated from Sunsik, a Korean ready-to-eat food prepared from grains, fruits, and vegetables. In this study, 39 unique B. cereus strains were isolated and identified from Sunsik samples, with an average contamination level of 10 to 200 CFU/g. The detection rates of the hblACD, cytK, and bceT genes among all the strains were 48.7, 66.7, and 87.1%, respectively. All 39 B. cereus strains carried nheABC and entFM genes, and 36 strains also had the ces gene, which encodes an emetic toxin. Nonhemolytic enterotoxin and hemolysin BL enterotoxin were produced by 39 and 26 strains, respectively. The strains were separated into 13 profiles based on the presence or absence of toxins and their genes, as determined by antibody tests and PCR analysis. Profile 1 was the largest group, comprising 30.7% (12 of 39) of the B. cereus strains tested; these strains harbored all toxins and their genes. The B. cereus strains were susceptible to most of the antibiotics tested but were highly resistant to b -lactam antibiotics. The repetitive element sequence polymorphism PCR fingerprints of the B. cereus strains were not influenced by the presence of toxin genes or antibiotic resistance profiles. Our results suggest that B. cereus strains from Sunsik could cause either the diarrheal or emetic types of food poisoning because all strains isolated contained at least one toxin and its gene, although the level of B. cereus contamination in Sunsik was low.     

Potential of Bacillus cereus for producing an emetic toxin,cereulide, in bakery products: quantitative analysis by chemical and biological methods

A method for the direct quantitative analysis of cereulide, the emetic toxin of Bacillus cereus, in bakery products was developed. The analysis was based on robotized extraction followed by quantitation of cereulide by liquid chromatography-mass spectrometry and an assay of toxicity by the boar sperm motility inhibition test. The bioassay and the chemical assay gave comparable results, demonstrating that the extracted cereulide was in a biologically active form. Cereulide was formed when cereulide-producing B. cereus strains were present at > or = 10(6) CFU/g in products with water activity values of > 0.953 and pHs of > 5.6. Rice-containing pastries accumulated high contents of cereulide (0.3 to 5.5 microg/g [wet weight]) when stored at nonrefrigeration temperatures (21 to 23 degrees C). Cereulide was not formed in products stored at refrigeration temperatures (4 to 8 degrees C). Cereulide is not inactivated by heating during food processing. Therefore, direct analysis of this toxin in food is preferable to cultivating methods for assessing the risk of food poisoning by emetic B. cereus.     

蜡样芽孢杆菌致吐毒素的研究现状

蜡样芽孢杆菌致吐毒素是热处理也不能灭活的毒素,没有方法可以去除食品中致吐毒素Cereulide。因此,探讨食品中哪些因素影响致吐毒素产生显得至关重要。本文对蜡样芽孢杆菌孢子、致吐毒素产生株的特点及致吐食源性中毒,分离、检测和定量食品中Cereulide的国内外新方法及影响致吐毒素产生的因素等进行了综述。     

Cases of emesis associated with bacterial contamination of an infant breakfast cereal product

A commercial product for infants containing cereal mixed with dried infant formula was diagnosed as producing rapid projectile vomiting in two infants. Analysis of multiple samples of the cereal product revealed significant contamination with two spore-forming species, Bacillus subtilis and a strain of Bacillus cereus. The latter is the most likely cause of the emetic food poisoning, but we were unable to detect B. cereus emetic toxin. This raises the possibility of the cause being either a new cereulide-type toxin, or the bacterial load, in which case the presence of B. subtilis could have been a contributing factor.     

蜡状芽孢杆菌(Bacillus celreus)污染及其对食品安全的影响

蜡状芽孢杆菌是常见的食品污染菌，在工业化社会中正成为日益重要的食物病原菌。它能产生一种呕吐毒素和多种肠毒素，主要引发呕吐和腹泻型食物中毒。与蜡状芽孢杆菌同属的苏云金杆菌也能产生类似的肠毒素，近来发现它可能与食品中毒有关。我国应当加强对蜡状芽孢杆菌的监测和研究。     

Food poisoning associated with pumilacidin-producing Bacillus pumilus in rice

Food poisoning caused by other Bacillus species than B. cereus has been described, but the toxins involved have rarely been isolated. Endospores will survive heat treatment and will germinate and multiply in cooked foods producing toxins under appropriate conditions. We describe a small food poisoning outbreak where three people became ill after a dinner in a Chinese restaurant. Acute symptoms including dizziness, headache, chills and back pain developed during the meal, and a few hours later they got stomach cramps and diarrhoea which lasted for several days. Cooked, reheated rice was the prime suspect of the food poisoning, and from the rice large numbers of Bacillus pumilus were isolated. The isolated B. pumilus strain was found to produce a complex of lipopeptides known as pumilacidins with the highest amounts produced at 15 degrees C. This is the first report on isolation of a pumilacidin-producing B. pumilus strain from food implicated in food poisoning and characterization of the organism and the toxin complex involved.     

云南边贸进口即食食品中蜡样芽胞杆菌污染状况及其呕吐毒素基因型检测

为探究云南省边贸进口即食食品中蜡样芽胞杆菌的污染状况及其呕吐毒素基因型携带情况,从云南边境各口岸进口食品贸易集散点(边民互市点、农贸市场以及进出口产品店等地)共采集市售包装样品224份。通过VITEK 2 compact全自动微生物鉴定系统和双重实时荧光聚合酶链式反应(Double real-time fluorescent polymerase chain reaction,Dual real-time PCR)技术对分离得到的蜡样芽胞杆菌进行鉴定,并对阳性菌株中的呕吐毒素基因进行检测。结果表明:蜡样芽胞杆菌在所采集样品中的检出率为20.09%(45/224),天保、金水河、瑞丽以及畹町各口岸检出率均高于10%。检出率较高的食品类别为:酱腌菜60%(3/5)、调味品50%(15/30)、水产及其制品46.67%(7/15);从45份阳性样品中共分离出113株蜡样芽胞杆菌,其中呕吐型毒素基因cesB的携带率为0.88%(1/113),呕吐型毒素基因携带率较低,但是蜡样芽胞杆菌的污染风险仍不可忽视。该研究结果对边贸进口食品安全、食源性疾病的监测及预防提供了理论依据。     

Toxin genes profiles and toxin production ability of Bacillus cereus isolated from clinical and food samples

Bacillus cereus can cause diarrheal and emetic type of food poisoning but little study has been done on the main toxins of food poisoning caused by B. cereus in Korea. The objective of this study is to characterize the toxin gene profiles and toxin-producing ability of 120 B. cereus isolates from clinical and food samples in Korea. The detection rate of nheABC, hblCDA, entFM, and cytK enterotoxin gene among all B. cereus strains was 94.2, 90.0, 65.8, and 52.5%, respectively. The ces gene encoding emetic toxin was not detected in all strains. Bacillus cereus strains carried at least 1 of the 8 enterotoxin genes were classified into 12 groups according to the presence or absence of 8 virulence genes. The 3 major patterns, I (nheABC, hblCDA, entFM, and cytK gene), II (nheABC, hblCDA and entFM gene), and VI (nheABC and hblCDA gene), accounted for 79.2% of all strains (95 out of 120 B. cereus isolates). Non-hemolytic enterotoxin (NHE) and hemolysin BL (HBL) enterotoxins were produced by 107 and 100 strains, respectively. Our finding revealed that NHE and HBL enterotoxins encoded by nhe and hbl genes were the major toxins among B. cereus tested in this study and enterotoxic type of B. cereus was predominant in Korea.     

Influence of temperature shifts on survival, growth, and toxin production by psychrotrophic and mesophilic strains of Bacillus cereus in potatoes and chicken gravy.

A study was done to determine the influence of temperature on growth and toxin production characteristics of psychrotrophic and mesophilic strains of Bacillus cereus when inoculated into mashed potatoes and chicken gravy containing various concentrations of sodium chloride and held at temperatures different from those at which cells had been cultured. Logarithmic growth phase cells (10 h, 30 degrees C) of psychrotrophic (F3802A/84) and mesophilic (B4ac-1) strains of Bacillus cereus were inoculated into rehydrated commercially processed instant mashed potatoes and chicken gravy supplemented with 0, 2, or 4% sodium chloride. Growth, survival, and diarrheal toxin production in potatoes and gravy held at 30, 37, and 10 degrees C (strain F3802A/84) or 30, 40, and 10 degrees C (strain B4ac-1) were monitored. Both strains grew in both foods containing no added sodium chloride or 2% sodium chloride when held at 30, 37, or 40 degrees C for 2 days. Strain B4ac-1 grew better than strain F3802A/84 in foods containing 4% sodium chloride. Maximum amounts of enterotoxin (1024 ng/g) were produced by strain B4ac-1 in chicken gravy held at 30 and 40 degrees C. Strain F3802A/84 grew to populations of 7 log10 CFU/g in foods containing no added sodium chloride or 2% sodium chloride at 10 degrees C. Strain F3802A/84 produced the highest amount of enterotoxin (1024 ng/g) at 30 degrees C in chicken gravy containing 0.7 or 2% sodium chloride; however, little or low amounts of toxin (4-16 ng/g) were produced in chicken gravy at 10 degrees C. Compared to strain B4ac-1, cells of strain F3802A/84 subjected to a downward shift in incubation temperature (10 degrees C) grew more rapidly in chicken gravy. Strain B4ac-1 produced the highest amount of toxin (1024 ng/g) at 30 degrees C in gravy containing 4% sodium chloride and at 40 degrees C in gravy containing 0.7% sodium chloride. Toxin was not detected in inoculated mashed potatoes. Results of this study indicate that shifts in incubation temperature influence growth and toxin production by psychrotrophic and mesophilic strains of B. cereux differently. It is important to store pasteurized, ready-to-eat foods at a temperature low enough to prevent the growth of B. cereus.     

Presence of Bacillus cereus in street foods in Gaborone, Botswana

The purpose of this study was to evaluate the microbiological safety and quality of street foods sold in Gaborone, Botswana. A total of 148 point-of-sale composite street food samples were bought and analyzed between June 2001 and May 2002. The analysis focused on the level of contamination of various street foods with Bacillus cereus. The B. cereus (vegetative and spores), total spore, and total viable counts were determined on all the samples. Also B. cereus isolates from 444 individual point-of-sale food samples were characterized with respect to their biochemical profiles and enterotoxigenic properties. The B. cereus contamination rate for point-of-sale foods was 65%. The B. cereus counts ranged from not detectable to levels as high as 9.1 log CFU/g. Despite the high rate of contamination of some samples, generally, most samples had B. cereus counts of less than 4 log CFU/g; hence, they were of acceptable microbiological quality. Bacillus diarrheal enterotoxin was detected from 52 isolates from individual portions of meals using the B. cereus enterotoxin reversed passive latex agglutination kit. Results of the assay revealed that 59.6% of the B. cereus isolates were enterotoxigenic. Most of the enterotoxigenic isolates were obtained from vegetable samples.     

Heat treatment for the control of Bacillus cereus spores in foods

The effect of heat treatments on food products related to soybean curd contaminated with Bacillus cereus spores was investigated for the purpose of preventing food-poisoning outbreaks by B. cereus. In the case of B. cereus strains isolated from foods, heating foods inoculated with the spores for 20 min at 70 degrees C, 5 min at 75 degrees C, 2 min at 100 degrees C, or 10 sec with a microwave oven plus reheating after standing for 2 hr at 25 degrees C reduced the number of surviving cells within the foods to less than one-hundredth. In the case of a heat-resistant strain isolated from a food poisoning outbreak, heating for 20 min at 70 degrees C plus reheating after standing for 1.5-2 hr at 35 degrees C, heating for 10 min at 75 degrees C plus reheating after standing for 1.5-2 hr at 35 degrees C, or heating for 2 min at 100 degrees C plus reheating after standing for 4 hr at 25 degrees C was effective. There was not much difference between the flavor components in foods with and without heat treatment at 70 degrees C, as analyzed by gas chromatography-mass spectrometry. These results indicate that these heat treatments are available to control B. cereus spores, without affecting the sensory quality of the foods.     

A PCR assay based on a sequence-characterized amplified region marker for detection of emetic Bacillus cereus

A PCR assay for the detection of Bacillus cereus strains able to produce an emetic toxin (cereulide) was developed in this study based on a sequence-characterized amplified region (SCAR) derived from a random amplified polymorphic DNA (RAPD) fragment. One of the RAPD fragments generated was selected, cloned, and sequenced. A set of PCR primers was newly designed from the SCAR obtained (the sequence of the cloned RAPD fragment) and used in this assay. To determine the specificity of the assay, 30 different B. cereus strains, 8 other Bacillus strains (of six species), and 16 other non-Bacillus strains (from 16 genera) were tested. Results were positive for every emetic B. cereus strain and for only one nonemetic B. cereus strain. For all other bacterial strains, results were negative. Bacterial DNA for PCR was prepared by a simple procedure using Chelex 100 resin from the bacterial colony on the agar plate or from culture after growth in brain heart infusion medium. This PCR assay enabled us to detect the bacteria of emetic B. cereus grown on agar plates but not the bacteria of nonemetic B. cereus. To test this PCR assay for the monitoring of the emetic bacteria, 10 to 70 CFU of B. cereus DSM 4312 (emetic) per g of food was inoculated into several foods as an indicator, followed by a 7-h enrichment culture step. Because this PCR assay based on the SCAR derived from the RAPD fragment was able to detect bacterial cells, this assay should be useful for rapid and specific detection of emetic B. cereus.     

Atmospheric oxygen and other conditions affecting the production of cereulide by Bacillus cereus in food

Factors influencing the production of cereulide, the emetic toxin of Bacillus cereus in food and laboratory media were investigated, using liquid chromatography-ion trap mass spectrometry and sperm motility inhibition bioassay for detection and quantitation. Oxygen was essential for production of the emetic toxin by B. cereus. When beans, rice or tryptic soy broth were inoculated with cereulide producing strains B203, B116 (recent food isolates) or the strain F-4810/72, high amounts (2 to 7 microg ml(-1) or g(-1) wet wt) of cereulide accumulated during 4-day storage at room temperature. In parallel cultures and foods, stored under nitrogen atmosphere (> 99.5% N2), less than 0.05 microg of cereulide ml(-1) or g(-1) wet wt accumulated. The outcome of the bioassay matched that of the chemical assay, with no indication of interference by substances in the rice or beans. Boiling for 20 to 30 min did not inactivate cereulide or cereulide producing strains in rice or the beans. Adding l-leucine and l-valine (0.3 g l(-1)) stimulated cereulide production 10- to 20-fold in R2A and in rice water agar. When the B. cereus strains were grown on agar media under permissive conditions (air, room temperature), cereulide was produced overnight with little or no increase when the incubation was extended to 4 days. In broth culture, the production of cereulide started later than 16-24 h. Anoxic storage prevented cereulide production also when the amino acids had been supplied. Packaging with modified atmosphere low in oxygen may thus be used to reduce the risk of cereulide formation during storage of food.     

Improved multiplex PCR assay for simultaneous detection of Bacillus cereus emetic and enterotoxic strains

Toxin producing Bacillus cereus can cause enterotoxic and/or emetic food poisoning. In the present study, a multiplex PCR assay was developed to detect all toxin genes known to be involved in food poisoning of B. cereus in a single reaction. Specific primers for the detection of enterotoxic (entFM, hblC, nheA, and cytK) genes and emetic toxin production (2 primer pairs: ces, CER) were designed based on the GeneBank sequences. The developed multiplex PCR assay was evaluated in pure culture and artificially inoculated milk, using 43 B. cereus strains and non-target strains. In brief, sensitivity in pure culture was 10-fold or more higher than artificially inoculated milk in multiplex PCR detection limit assay. The presented PCR assay is a developed molecular tool for the rapid simultaneous detection of emetic and enterotoxin producing B. cereus strains.     

Computer aided boar semen motility analysis for cereulide detection in different food matrices

Computer Aided Semen Analysis (CASA) study of the boar semen motility has been demonstrated to be an appropriate assay for detection of cereulide (Bacillus cereus emetic toxin). Application of the boar semen bio-assay to detect cereulide directly in foods requires investigation of potential interference of food components, preservatives and other microbial and chemical food contaminants with the bio-assay. Current study provides evidence that none of included Staphylococcus aureus enterotoxins A, B, C and D nor B. cereus Hemolysin BL (HBL) and non-hemolytic enterotoxin (NHE) and three mycotoxins (Sterigmatocystin, Fumonisin B1 and Patulin) exhibited a toxic impact on semen progressive motility. Aflatoxin M1, M3 and zearalenone impaired semen motility only at concentrations (0.004 mg ml(-1), 0.1 mg ml(-1) and 10 mg ml(-1), respectively) much higher than those found in foods and those permitted by legislation, in comparison to cereulide which induces motility cease at concentrations lower than 20 ng ml(-1). Ten commonly used preservatives, namely potassium sorbate, sodium benzoate, (DL) malic acid, citric acid, (L+) tartaric acid, acetic acid, (DL) lactic acid, (L+) ascorbic acid, sodium chloride and sucrose induced no cease in spermatozoa motility even at preservative concentrations higher than permitted by legislation. Dioxins, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and acrylamide had no acute effect on spermatozoa motility at concentrations of 500 and 10,000 mg ml(-1), respectively. Robustness of computer aided boar semen motility analysis, tested with 14 different foods inoculated with cereulide producing B. cereus, showed distinct cereulide production in seven samples (although B. cereus growth to counts higher than 8 log CFU g(-1) was noted in 11 samples), in amounts close to those reported in foodborne outbreaks. Test evaluation in 33 samples suspected to hold cereulide showed actual cereulide presence in ten samples and no interference of food matrix with the assay.     

基于qPCR快速检测米饭中产呕吐毒素蜡样芽孢杆菌的研究

为了建立基于QPCR的快速方法检测米饭中产呕吐毒素蜡样芽孢杆菌,首先采用煮沸法提取蜡样芽孢杆菌基因组DNA,并利用普通PCR方法验证引物特异性,然后通过在米饭样品中添加目标菌,模拟受污染的实际样本,用QPCR技术定量检测米饭中产呕吐毒素蜡样芽孢杆菌。结果表明该方法具有快速、特异性强、灵敏度高和稳定性好的优点,能对产呕吐毒素蜡样芽孢杆菌定量。不经过增菌培养,实际样品的检测限为9.8×101CFU/g;经过2 h的增菌培养,检测限能达到100CFU/g;并且米饭中添加其他杂菌后,不影响对蜡样芽孢杆菌的检测。建立的QPCR方法适用于米饭等相关淀粉类食品中产呕吐毒素蜡样芽孢杆菌的检测,从而为监测该菌所致食品污染以及早期相关食物中毒提供快速定量检测方法。     

Establishment of a novel multiplex PCR assay and detection of toxigenic strains of the species in the Bacillus cereus group

Five different enterotoxins and one emetic toxin of Bacillus cereus have been characterized. To amplify all of the enterotoxin and emetic-specific sequences of the species in the B. cereus group, a multiplex PCR with 12 primer pairs was established. In developing the assay method, a common terminal sequence at the 3' ends of all primers was chosen and a hot start Taq polymerase was used to overcome primer dimer formation. The assay was successfully applied to analyze the toxigenic potential of 162 food-poisoning and food-related strains. Results showed that there were 10 toxigenic patterns for all the test strains. All of the B. cereus strains carried at least one toxin gene. More than 70% of Bacillus mycoides strains carried no known toxin genes. The toxin profiles and toxin genes of B. mycoides strains were significantly different from B. cereus strains (P < 0.05), although the two species were closely related. The results suggest that many B. mycoides strains might be less prone to cause food poisoning. They also indicate the importance of detecting the toxin genes together with the detection of the species in the B. cereus group.