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Members of the B. cereus group are spore-forming organisms commonly associated with spoilage of milk and dairy products. We have determined the genetic identity and growth characteristics of 57 B. cereus isolates collected from a Norwegian ice cream production plant. Our findings revealed persistence of B. cereus spp. strains for up to 19 months, suggesting the plant's susceptibility to long-term colonization. One of the mesophilic isolates, NVH-YM303, carried a complete cereulide synthetase operon. To assess the potential food poisoning risk associated with the presence of cereulide-producing strains in the production line, we examined the production of cereulide in ice cream and milk at different temperatures by NVH-YM303 and by the emetic psychrotrophic B. weihenstephanensis strain BtB2-4. Our findings revealed that NVH-YM303 produced higher levels of cereulide in ice cream as compared to milk. Furthermore, it was observed that NVH-YM303 produced more cereulide in ice cream at 25 °C compared to 15 °C. Conversely, BtB2-4 produced more cereulide in ice cream at 15 °C than at 25 °C. The results obtained in this study contribute to knowledge important for risk assessment of the potential hazards posed by the presence of B. cereus within ice cream production facilities. [ABSTRACT FROM AUTHOR]

Bacillus cereus is an emerging food-borne pathogen responsible for two types of food poisoning: emetic and diarrhoeal type. Here we report an emetic type of food-borne illness attributable to Bacillus cereus. On 2nd February, 2021, 202 people suffered from pain in abdomen and vomiting after consuming the rice provided during a public gathering in Diphu, Assam. Culture of leftover fried rice showed growth of Bacillus cereus group of organisms. Molecular detection of enterotoxin and emetic toxin genes was done in the isolated strains by polymerase chain reaction. Multi locus sequence typing (MLST) and phylogenetic analysis was done to characterise the isolated strains. A total of five strains of Bacillus cereus were isolated. Ces gene was found in isolates GMC22 & GMC24 and other enterotoxins producing genes were found in isolates GMC23 and GMC24. MLST identified four sequence types (STs) (ST1051, ST1616, ST998 and ST1000). Phylogenetic analysis clustered ST-1051 assigned to the GMC22 strain into the previously defined clade I and was in close relation with ST-144, representing a new cereulide-producing emetic cluster. As Bacillus cereus is a common contaminant of foods, it is essential to evaluate the pathogenic potential of the bacteria for a definite link between causative agents and the illness. MLST can be used to characterize the Bacillus cereus strains isolated from outbreak samples in order to link the probable pathogens with the illness. In this outbreak, we suggest that ST-1051 is the strain responsible for the food-borne illness, which was predominantly of emetic type. [ABSTRACT FROM AUTHOR]

Food intoxications evoked by emetic Bacillus cereus strains constitute a serious threat to public health, leading to emesis and severe organ failure. The emetic peptide toxin cereulide, assembled by the non-ribosomal peptide synthetase CesNRPS, cannot be eradicated from contaminated food by usual hygienic measures due to its molecular size and structural stability. Next to cereulide, diverse chemical variants have been described recently that are produced concurrently with cereulide by CesNRPS. However, the contribution of these isocereulides to the actual toxicity of emetic B. cereus, which produces a cocktail of these toxins in a certain ratio, is still elusive. Since cereulide isoforms have already been detected in food remnants from foodborne outbreaks, we aimed to gain insights into the composition of isocereulides and their impact on the overall toxicity of emetic B. cereus. The amounts and ratios of cereulide and isocereulides were determined in B. cereus grown under standard laboratory conditions and in a contaminated sample of fried rice balls responsible for one of the most severe food outbreaks caused by emetic B. cereus in recent years. The ratios of variants were determined as robust, produced either under laboratory or natural, food-poisoning conditions. Examination of their actual toxicity in human epithelial HEp2-cells revealed that isocereulides A-N, although accounting for only 10% of the total cereulide toxins, were responsible for about 40% of the total cytotoxicity. An this despite the fact that some of the isocereulides were less cytotoxic than cereulide when tested individually for cytotoxicity. To estimate the additive, synergistic or antagonistic effects of the single variants, each cereulide variant was mixed with cereulide in a 1:9 and 1:1 binary blend, respectively, and tested on human cells. The results showed additive and synergistic impacts of single variants, highlighting the importance of including not only cereulide but also the isocereulides in routine food and clinical diagnostics to achieve a realistic toxicity evaluation of emetic B. cereus in contaminated food as well as in patient samples linked to foodborne outbreaks. Since the individual isoforms confer different cell toxicity both alone and in association with cereulide, further investigations are needed to fully understand their cocktail effect. [ABSTRACT FROM AUTHOR]

Members of the B. cereus group are spore-forming organisms commonly associated with spoilage of milk and dairy products. We have determined the genetic identity and growth characteristics of 57 B. cereus isolates collected from a Norwegian ice cream production plant. Our findings revealed persistence of B. cereus spp. strains for up to 19 months, suggesting the plant’s susceptibility to long-term colonization. One of the mesophilic isolates, NVH-YM303, carried a complete cereulide synthetase operon. To assess the potential food poisoning risk associated with the presence of cereulide-producing strains in the production line, we examined the production of cereulide in ice cream and milk at different temperatures by NVH-YM303 and by the emetic psychrotrophic B. weihenstephanensis strain BtB2-4. Our findings revealed that NVH-YM303 produced higher levels of cereulide in ice cream as compared to milk. Furthermore, it was observed that NVH-YM303 produced more cereulide in ice cream at 25 °C compared to 15 °C. Conversely, BtB2-4 produced more cereulide in ice cream at 15 °C than at 25 °C. The results obtained in this study contribute to knowledge important for risk assessment of the potential hazards posed by the presence of B. cereus within ice cream production facilities.

Food intoxications evoked by emetic Bacillus cereus strains constitute a serious threat to public health, leading to emesis and severe organ failure. The emetic peptide toxin cereulide, assembled by the non-ribosomal peptide synthetase CesNRPS, cannot be eradicated from contaminated food by usual hygienic measures due to its molecular size and structural stability. Next to cereulide, diverse chemical variants have been described recently that are produced concurrently with cereulide by CesNRPS. However, the contribution of these isocereulides to the actual toxicity of emetic B. cereus, which produces a cocktail of these toxins in a certain ratio, is still elusive. Since cereulide isoforms have already been detected in food remnants from foodborne outbreaks, we aimed to gain insights into the composition of isocereulides and their impact on the overall toxicity of emetic B. cereus. The amounts and ratios of cereulide and isocereulides were determined in B. cereus grown under standard laboratory conditions and in a contaminated sample of fried rice balls responsible for one of the most severe food outbreaks caused by emetic B. cereus in recent years. The ratios of variants were determined as robust, produced either under laboratory or natural, food-poisoning conditions. Examination of their actual toxicity in human epithelial HEp2-cells revealed that isocereulides A-N, although accounting for only 10% of the total cereulide toxins, were responsible for about 40% of the total cytotoxicity. An this despite the fact that some of the isocereulides were less cytotoxic than cereulide when tested individually for cytotoxicity. To estimate the additive, synergistic or antagonistic effects of the single variants, each cereulide variant was mixed with cereulide in a 1:9 and 1:1 binary blend, respectively, and tested on human cells. The results showed additive and synergistic impacts of single variants, highlighting the importance of including not only cereulide but also the isocereulides in routine food and clinical diagnostics to achieve a realistic toxicity evaluation of emetic B. cereus in contaminated food as well as in patient samples linked to foodborne outbreaks. Since the individual isoforms confer different cell toxicity both alone and in association with cereulide, further investigations are needed to fully understand their cocktail effect.

This study analyzed the virulence, growth characteristics, and cytotoxicity of Bacillus cereus strains isolated from fresh produce, including romaine lettuce, sesame leaf, tomato, and cucumber grown by different methods. Polymerase chain reaction (PCR) was used to assess the toxigenic potential, and the cytotoxicity of B. cereus was estimated using cell-free supernatant in HEp-2 cells. The study found that hblD was the predominant diarrheal enterotoxin in the 59 isolated B. cereus strains, followed by nheB and hblC. The optimal temperatures for growth ranged from 42 to 44 °C, with the highest growth rates and shortest lag times. Cytotoxicity varied greatly depending on abiotic factors, including NaCl, pH, and medium, and was not always correlated with cell population. The study highlights the importance of establishing control measures to prevent B. cereus intoxication in fresh vegetables. [ABSTRACT FROM AUTHOR]

Cereulide, which can be produced by Bacillus cereus, is strongly associated with emetic-type food poisoning outbreaks. It is an extremely stable emetic toxin, which is unlikely to be inactivated by food processing. Considering the high toxicity of cereulide, its related hazards raise public concerns. A better understanding of the impact of B. cereus and cereulide is urgently needed to prevent contamination and toxin production, thereby protecting public health. Over the last decade, a wide range of research has been conducted regarding B. cereus and cereulide. Despite this, summarized information highlighting precautions at the public level involving the food industry, consumers and regulators is lacking. Therefore, the aim of the current review is to summarize the available data describing the characterizations and impacts of emetic B. cereus and cereulide; based on this information, precautions at the public level are proposed. [ABSTRACT FROM AUTHOR]

This study describes an emetic food-borne intoxication associated with a Bacillus cereus group species and the characterization of the bacterial isolates from the incident in aspects of molecular tying, genetic factors, cytotoxicity, and pathogenic mechanisms relating to emetic illness. Through the polyphasic identification approach, all seven isolates obtained from food and clinical samples were identified as Bacillus thuringiensis. According to multilocus sequence typing (MLST) analysis, intraspecific diversity was found within the B. thuringiensis isolates. Four allelic profiles were found, including two previously known STs (ST8 and ST15) and two new STs (ST2804 and ST2805). All isolates harbored gene fragments located in the cereulide synthetase (ces) gene cluster. The heat-treated culture supernatants of three emetic B. thuringiensis isolates, FC2, FC7, and FC8, caused vacuolation and exhibited toxicity to Caco-2 cells, with CC50 values of 56.57, 72.17, and 79.94 µg/mL, respectively. The flow cytometry with the Annexin V/PI assay revealed both apoptosis and necrosis mechanisms, but necrosis was the prominent mechanism that caused Caco-2 cell destruction by FC2, the most toxic isolate. [ABSTRACT FROM AUTHOR]

Cereulide, which can be produced by Bacillus cereus, is strongly associated with emetic-type food poisoning outbreaks. It is an extremely stable emetic toxin, which is unlikely to be inactivated by food processing. Considering the high toxicity of cereulide, its related hazards raise public concerns. A better understanding of the impact of B. cereus and cereulide is urgently needed to prevent contamination and toxin production, thereby protecting public health. Over the last decade, a wide range of research has been conducted regarding B. cereus and cereulide. Despite this, summarized information highlighting precautions at the public level involving the food industry, consumers and regulators is lacking. Therefore, the aim of the current review is to summarize the available data describing the characterizations and impacts of emetic B. cereus and cereulide; based on this information, precautions at the public level are proposed.

Bacillus cereus is a food-borne pathogenic bacterium that can cause infection and intoxication to human beings. Its ability to form spores and produce toxins are significant contributory factors to making it a great health risk for the consumer. This paper aims to provide an overview of the occurrence of emetic and diarrhoeal food poisoning caused by B. cereus in Southeast Asia. It concerns foods commonly consumed by Southeast Asia citizens, such as fresh food, beverages and traditional food. Rice is the food most associated with B. cereus contamination. Methods used in detecting and quantifying B. cereus and enterotoxins as well as cereulides are compiled in this paper. Bacillus cereus can be identified using biochemical tests or commercially available kit. The methods used to detect the emeticproducing B. cereus are HEp-2 cell vacuole formation, polymerase chain reaction (PCR), commercial kit and High-Performance Liquid Chromatography (HPLC). On the other hand, diarrhoeal-producing B. cereus can be detected using a commercial kit and real-time PCR. The food safety laws and regulations implemented in Southeast Asian countries are also included and precautionary steps are suggested. Food poisoning due to B. cereus is always overlooked because it has a short duration of illness and the symptoms are usually mild. [ABSTRACT FROM AUTHOR]

Probiotic bacteria mainly Lactic Acid Bacteria (LAB) demonstrate to equilibrate the intestinal microflora. Strains of the Bacillus species have been extensively used as probiotics for human beings, animal feed as well as plant promoting bacteria. Attempts were made to characterize and assess the safety of Bacillus strains for their putative virulence factors. The present study includes screening the isolates by hemolysis on blood agar, DNase, lecithinase and cytotoxic activity as well as detection of toxins in the culture medium. PCR technique was used to detect genes encoding enterotoxin T (bceT), cytotoxin K (CytK), enterotoxin FM (EntFM), non-hemolytic enterotoxin (NheA, NheB), haemolysin BL (HblA, HblC, HblD) and emetic toxin (ces). Among seven Bacillus isolates, only isolates B. cereus sp. M14, B. subtilis sp. M12 and B. methylotrophicus sp. M8 showed weak hemolysis on blood agar. DNase and lecithinase activity were not observed for all isolates. However, isolates B. subtilis sp. M12 and B. cereus sp. M14 indicated low cytotoxic effects on HepG2 cell line. Using two commercial immunoassay kits, no enterotoxin was detected for all isolates. Nhe and Hbl genes were detected in isolates B. methylotrophicus sp. M8 and B. cereus sp. M14, respectively. In contrast, none of the Bacillus isolates harbored bceT, entFM and ces genes in their chromosomes. Taking together, among the evaluated Bacillus isolates, strains B. pumilus sp. M17 and B. pumilus sp. S10 found to be the most promising candidates to fulfill the safety assessments of putative virulence factors suitable for human consumption. [ABSTRACT FROM AUTHOR]

This study aims to investigate the potential role of lactose on cereulide biosynthesis by emetic Bacillus cereus in dairy matrices. The cereulide yields in whole milk and lactose-free milk were investigated using the emetic reference strain F4810/72. To eliminate the influence of complex food substrates, the LB medium model was further used to characterize the effect of lactose on cereulide produced by F4810/72 and five other emetic B. cereus strains. Results showed that the lactose-free milk displayed a 13-fold higher amount of cereulide than whole milk, but the cereulide level could be reduced by 91 % when the lactose content was restored. The significant inhibition of lactose on cereulide yields of all tested B. cereus strains was observed in LB medium, showing a dose-dependent manner with inhibition rates ranging of 89–98 %. The growth curves and lactose utilization patterns of all strains demonstrated that B. cereus cannot utilize lactose as a carbon source and lactose might act as a signal molecule to regulate cereulide production. Moreover, lactose strongly repressed the expression of cereulide synthetase genes (ces), possibly by inhibiting the key regulator Spo0A at the transcriptional level. Our findings highlight the potential of lactose as an effective strategy to control cereulide production in food. [Display omitted] • Lactose significantly inhibited cereulide yields of emetic B. cereus in milk products. • Lactose might act as a signal molecule. • Lactose repressed the ces genes expression at the transcriptional level. • Expression of ces genes was possibly regulated by the spo0A transcription. [ABSTRACT FROM AUTHOR]

Graphical abstract Abstract Homocereulide, isolated from marine bacterium Bacillus cereus , is an analog of emetic toxin cereulide. There is no report on its structure determination and involvement in B. cereus -associated food poisoning. Homocereulide is a cyclic dodecadepsipeptide composed of l - O -Val- l -Val- d - O -Leu- d -Ala and l - O - allo -Ile- d -Val- d - O -Leu- d -Ala. Here, we synthesized homocereulide using liquid phase fragment condensation. The NMR spectrum of synthesized homocereulide confirmed the intended structure and LC-MS results were consistent with natural products. Morphological evaluation using HEp-2 cells showed higher toxicity with homocereulide (1.39 nM) than cereulide (3.95 nM). Though cereulide is the main component in broth culture, homocereulide is also likely involved in B. cereus -associated food poisoning. [ABSTRACT FROM AUTHOR]

Bacillus cereus is a human pathogenic bacterium found in foods with the potential to cause emesis and diarrhea. This study estimated the presence, toxigenic and genomic diversity of B. cereus s.l. obtained from cassava starch samples collected in bakeries and powdered food companies in Medellín (Colombia). Bacillus cereuss.l. was found in 43 of 75 (57%) cassava starch samples and 98 isolates were obtained. The nheABC, hblCDAB, cytK2, entFM and cesB toxin genes were detected by multiplex PCR and the most frequent operon was nheABC, whereas cesB gene was not found. Twelve toxigenic profiles were determined by the detection of toxin genes, and the most frequent profiles harbored all enterotoxin genes. A broad genomic diversity was detected according to GTG5-PCR fingerprinting results with 76 B. cereus s.l. grouped in sixteen clusters and the 22 isolates clustering separately. No relationship was observed between genomic background and toxigenic profiles. In general, the results showed a high genomic and enterotoxigenic diversity in B. cereus s.l. found in cassava starch. These results should incentive future studies to understand the distribution of B. cereus s.l. isolated on raw materials in comparison with finished products.

Resumen Bacillus cereus es una bacteria genéticamente diversa que se encuentra comúnmente en el ambiente. Contamina los alimentos afectando la salud humana, al ingerir el microorganismo y/o sus toxinas, la emética o las enterotoxinas. En Colombia son escasos los reportes de intoxicación por B. cereus y se estima que hay un gran subregistro. Por lo anterior, se recomienda aumentar la vigilancia de este patógeno y realizar estudios sobre aspectos relevantes que permitan aplicar medidas de control para disminuir las intoxicaciones por B. cereus. El objetivo de esta revisión bibliográfica es presentar información actualizada sobre B. cereus, que incluye aspectos de su biología, taxonomía, toxinas, alimentos que contamina y metodologías para detectar, prevenir y controlar este microorganismo. La información presentada es de utilidad para el público en general, especialmente personas vinculadas al sector de alimentos, inocuidad alimentaria y control de procesos. / Abstract Bacillus cereus is a genetically diverse bacterium commonly found in the environment. It contaminates food, thus affecting human health upon ingestion of the microorganism and/or its toxins, the emetic or enterotoxins. In Colombia, reports of intoxication by B. cereus are scarce and under-registration is presumed. Because of this, it is recommended to increase surveillance of this pathogen and to develop studies on relevant aspects that allow the application of control measures to reduce intoxications by B. cereus. The aim of this review is to present current information on B. cereus, including aspects of its biology, taxonomy, toxins, food that it contaminates and methodologies for the detection, prevention and control of this microorganism. This information is useful for the general public, especially people involved with the food sector, food safety and process control.

Potential pathogens of foodborne toxic infections – bacterial contaminants Bacillus cereus isolated from plant raw materials and food products from the Ukrainian region were investigated. When determining of the proportion of isolated bacilli from the plant samples, it was established that the epidemiologically significant microorganisms of Bacillus cereus as agents of food poisoning are the second largest. The average value of contaminated samples of Ukrainian plant raw materials and processed products with Bacillus cereus is 36,2 %. The ability of Bacillus cereus strains identified by a complex of morphological, tinctorial, cultural and biochemical properties, to produce specific emetic and enterotoxins was studied. Molecular genetic diagnosis and detection of the toxin-producing ability of isolated 42 Bacillus cereus strains showed both the possibility of their rapid identification and the presence of specific toxicity genes. Multiplex polymerase chain reaction (PCR) was carried out with specific primers to detect toxicity determined of various bacilli genes: nheA, hblD, cytK, cesВ. The distribution of toxigenic genes is significantly different among the Bacillus cereus isolates from various sources. The nheA, hblD and cytK enterotoxin genes were detected in 100, 83,3 and 61,9 % of the investigated strains of Bacillus cereus, respectively. The cesB gene encoding emetic toxin was detected in 4,8 % of strains. Molecular-genetic PCR-method confirmed that all the isolated strains belong to the Bacillus cereus group, and the ability to produce toxins can be attributed to five groups. The main toxins that produce the investigated Bacillus cereus strains were nhe and hbl enterotoxins encoded by the corresponding genes of nheA and hblD. The enterotoxic type of Bacillus cereus was predominant in Ukrainian region. Studies of domestic plant food raw materials and products have confirmed the need to improve microbiological control of product safety by introducing accelerated specific diagnostics of contaminants by molecular genetics methods.

Cereulide (CER) intoxication occurs at relatively high doses of 8 µg/kg body weight. Recent research demonstrated a wide prevalence of low concentrations of CER in rice and pasta dishes. However, the impact of exposure to low doses of CER has not been studied before. In this research, we investigated the effect of low concentrations of CER on the behavior of intestinal cells using the Caco-2 cell line. The MTT (mitochondrial 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and the SRB (sulforhodamine B) reactions were used to measure the mitochondrial activity and cellular protein content, respectively. Both assays showed that differentiated Caco-2 cells were sensitive to low concentrations of CER (in a MTT reaction of 1 ng/mL after three days of treatment; in an SRB reaction of 0.125 ng/mL after three days of treatment). Cell counts revealed that cells were released from the differentiated monolayer at 0.5 ng/mL of CER. Additionally, 0.5 and 2 ng/mL of CER increased the lactate presence in the cell culture medium. Proteomic data showed that CER at a concentration of 1 ng/mL led to a significant decrease in energy managing and H2O2 detoxification proteins and to an increase in cell death markers. This is amongst the first reports to describe the influence of sub-emetic concentrations of CER on a differentiated intestinal monolayer model showing that low doses may induce an altered enterocyte metabolism and membrane integrity.

Bacillus cereus is a food contaminant and a known human pathogen that can cause emetic and diarrheal syndromes. In this study we evaluated the presence of toxigenic B. cereus by multiplex PCR directly in dietary complement for children and cassava starch samples collected on Medellin, Colombia. Of 75 dietary complement for children samples evaluated, 70.7% were contaminated with toxigenic B. cereus and four different toxigenic consortia were detected: I: nheA, hblC, cytK (9.8%), II: nheA, hblC (2%), III: hblC, cytK (41.2%), IV: hblC (47%). Of 75 cassava starch samples, 44% were contaminated with toxigenic B. cereus and four different toxigenic consortia were determined: I: nheA, hblC, cytK (48.5%), II: nheA, hblC, cytK, cesB (3%), III: hblC, cytK (30.3%), IV: hblC (18.2%). In general, in dietary complement for children only enterotoxigenic consortia were detected while in cassava starch the enterotoxigenic consortia predominated over the emetic. Multiplex PCR was useful to detect toxigenic B. cereus contamination allowing direct and imultaneous detection of all toxin genes in foods. This study is the first in Colombia to evaluate toxigenic B. cereus, providing information of importance for microbiological risk evaluation in dried foods.

Besides Bacillus cereus , some strains of the psychrotolerant, potentially foodborne pathogen Bacillus weihenstephanensis can produce the emetic toxine (cereulide). This toxin is a heat- and acid-stable cyclic dodecadepsipeptide that causes food intoxication with vomiting. However, some severe clinical cases with lethal outcomes have been described. If cereulide can be produced during refrigerated storage, it will not be inactivated by reheating food, representing an important risk of food intoxication for consumers. In this paper, we determined the capacity of the B. weihenstephanensis strains BtB2-4 and MC67 to grow and produce cereulide on agar media at temperatures from 8 °C to 25 °C and at a pH from 5.4 to 7.0. At 8 °C, strain BtB2-4 produced quantifiable amounts of cereulide, whereas the limit of detection was reached for strain MC67. For BtB2-4, cereulide production increased 5-fold between 8 °C and 10–15 °C and by more than 100-fold between 15 °C and 25 °C. At temperatures of 10 °C and higher, cereulide concentrations were within the range of those reported by previous works in foods implicated in emetic poisoning. At 25 °C, decreasing the pH to 5.4 reduced cereulide production by strain BtB2-4 by at least 20-fold. [ABSTRACT FROM AUTHOR]

Copyright of Food Science & Technology (2073-8684) is the property of Odesa National University of Technology and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

In recent years, the emetic toxin cereulide, produced by Bacillus cereus, has gained high relevance in food production and food safety. Cereulide is synthesized nonribosomal by the multi-enzyme complex Ces-NRPS, which is encoded on a megaplasmid that shares its backbone with the Bacillus anthracis pX01 toxin plasmid. Due to its resistance against heat, proteolysis and extreme pH conditions, the formation of this highly potent depsipeptide toxin is of serious concern in food processing procedures including slow cooling procedures and /or storage of intermediate products at ambient temperatures. So far, systematic data on the effect of extrinsic factors on cereulide synthesis has been lacking. Thus, we investigated the influence of temperature, a central extrinsic parameter in food processing, on the regulation of cereulide synthesis on transcriptional, translational and post-translational levels over the growth temperature range of emetic B. cereus. Bacteria were grown in 3°C interval steps from 12°C to 46°C and cereulide synthesis was followed from ces gene transcription to cereulide toxin production. This systematic study revealed that temperature is a cardinal parameter, which primarily impacts cereulide synthesis on post-transcriptional levels, thereby altering the composition of cereulide isoforms. Our work also highlights that the risk of cereulide production could not be predicted from growth parameters or sole cell numbers. Furthermore, for the first time we could show that the formation of the recently identified cereulide isoforms is highly temperature dependent, which may have great importance in terms of food safety and predictive microbiology. Notably the production of isocereulide A, which is about 10-fold more cytotoxic than cereulide, was specifically supported at low temperatures.

The emetic syndrome of Bacillus cereus is a food intoxication caused by cereulide (CER) and manifested by emesis, nausea and in most severe cases with liver failure. While acute effects have been studied in the aftermath of food intoxication, an exposure to low doses of cereulide might cause unnoticed damages to the intestines and liver. The toxicity which relies on the mitochondrial dysfunction was assessed on Caco-2 and HepG2 cells after exposure of one, three and ten days to a range of low doses of cereulide. Oxygen consumption rate analyses were used to study the impact of low doses of CER on the bioenergetics functions of undifferentiated Caco-2 and HepG2 cells using Seahorse XF extracellular flux analyzer. Both Caco-2 and HepG2 cells experienced measurable mitochondrial impairment after prolonged exposure of 10 days to 0.25 nM of cereulide. Observed mitochondrial dysfunction was greatly reflected in reduction of maximal cell respiration. At 0.50 nM CER, mitochondrial respiration was almost completely shut down, especially in HepG2 cells. These results corresponded with a severe reduction in the amount of cells and an altered morphology, observed by microscopic examination of the cells. Accurate and robust quantification of basal respiration, ATP production, proton leak, maximal respiration, spare respiratory capacity, and non-mitochondrial respiration allowed better understanding of the effects of cereulide in underlying respiratory malfunctions in low-dose exposure.

Bacillus cereus is a gram-positive endospore forming bacterium known for its wide spectrum of phenotypic traits, enabling it to occupy diverse ecological niches. Although the population structure of B. cereus is highly dynamic and rather panmictic, production of the emetic B. cereus toxin cereulide is restricted to strains with specific genotypic traits, associated with distinct environmental habitats. Cereulide is an ionophoric dodecadepsipeptide that is produced non-ribosomally by an enzyme complex with an unusual modular structure, named cereulide synthetase (Ces NRPS). The ces gene locus is encoded on a mega virulence plasmid related to the Bacillus anthracis toxin plasmid pXO1. Cereulide, a highly thermo- and pH- resistant molecule, is preformed in food, evokes vomiting a few hours after ingestion and was shown to be the direct cause of gastroenteritis symptoms; occasionally it is implicated in severe clinical manifestations including acute liver failures. Control of toxin gene expression in emetic Bacillus cereus involves central transcriptional regulators, such as CodY and AbrB, thereby inextricably linking toxin gene expression to life cycle phases and specific conditions, such as the nutrient supply encountered in food matrices. While in recent years considerable progress has been made in the molecular and biochemical characterization of cereulide toxin synthesis, far less is known about the embedment of toxin synthesis in the life cycle of B. cereus. Information about signals acting on toxin production in the food environment is literally lacking. We summarize the data available on the complex regulatory network controlling cereulide toxin synthesis, discuss the role of intrinsic and extrinsic factors acting on toxin biosynthesis in emetic B. cereus and stress how unraveling these processes can lead to the development of novel effective strategies to prevent toxin synthesis in the food production and processing chain.

Copyright of Revista Facultad Nacional de Salud Pública is the property of Universidad de Antioquia, Facultad Nacional de Salud Publica and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

This study determined the prevalence and toxin profile of Bacillus cereus and Bacillus thuringiensis in doenjang, a fermented soybean food, made using both traditional and commercial methods. The 51 doenjang samples tested were broadly contaminated with B. cereus; in contrast, only one sample was positive for B. thuringiensis. All B. cereus isolates from doenjang were positive for diarrheal toxin genes. The frequencies of nheABC and hblACD in traditional samples were 22.7 and 0%, respectively, whereas 5.1 and 5.1% of B. cereus isolates from commercial samples possessed nheABC and hblACD, respectively. The detection rate of ces gene was 10.8%. The predominant toxin profile among isolates from enterotoxigenic B. cereus in doenjang was profile 4 (entFM-bceT-cytK). The major enterotoxin genes in emetic B. cereus were cytK, entFM, and nheA genes. The B. thuringiensis isolate was of the diarrheagenic type. These results provide a better understanding of the epidemiology of the enterotoxigenic and emetic B. cereus groups in Korean fermented soybean products. [ABSTRACT FROM AUTHOR]

The emetic toxin cereulide, which can be produced by Bacillus cereus, can be the cause of food poisoning upon ingestion by the consumer. The toxin causes vomiting and is mainly produced in farinaceous food products. This article includes the prevalence of B. cereus and of cereulide in food products in The Netherlands, a characterization of B. cereus isolates obtained, cereulide production conditions, and a comparison of consumer exposure estimates with those of a previous exposure assessment. Food samples (n = 1,489) were tested for the presence of B. cereus; 5.4% of the samples contained detectable levels (>10² CFU/ g), and 0.7% contained levels above 105 CFU/g. Samples (n = 3,008) also were tested for the presence of cereulide. Two samples (0.067%) contained detectable levels of cereulide at 3.2 and 5.4 µ/kg of food product. Of the 481 tested isolates, 81 produced cereulide and/or contained the ces gene. None of the starch-positive and hbl-containing isolates possessed the ces gene, whereas all strains contained the nhe genes. Culture of emetic B. cereus under nonoptimal conditions revealed a delay in onset of cereulide production compared with culture under optimal conditions, and cereulide was produced in all cases when B. cereus cells had been in the stationary phase for some time. The prevalence of cereulide-contaminated food approached the prevalence of contaminated products estimated in an exposure assessment. The main food safety focus associated with this pathogen should be to prevent germination and growth of any B. cereus present in food products and thus prevent cereulide production in foods. [ABSTRACT FROM AUTHOR]

Bacillus species are used in animal production directly as microbial feed additives or as the source of other feed additives, notably enzymes. The principal safety concern for consumers and, to a lesser extent livestock, associated with Bacillus is a capacity for toxin production. However, the capacity for toxin production and the nature of the toxins produced is unevenly distributed over the genus, occurring frequently in some species and more rarely in others. In principle, the selection of strains belonging to the B. cereus taxonomic group for direct use in animal production is considered inadvisable. If, however, they are proposed then the full genome should be sequenced and a bioinformatic analysis made to search for genes coding for enterotoxins and cereulide synthase. If there is evidence of homology, the non-functionality of the genes (e.g. mutation, deletion) must be demonstrated. For other species, concerns appear to be associated to the production of surfactin like-lipopeptides, although the relation between the presence of these compounds and/or other toxic factors and the risk of illness in human has not yet been established. In the absence of animal models shown to be able to distinguish hazardous from non hazardous strains, the FEEDAP Panel relies on the use of in vitro cell-based methods to detect evidence of a cytotoxic effect. Such tests should be made with culture supernatants since the concentration of cells obtained in a broth culture would always exceed that found in animal food products. If the strain proves to be cytotoxic it is not recommended for use.

Bacillus cereus can cause emetic and diarrheal food poisoning. It is widespread in nature and therefore, considered a major foodborne pathogen. To develop a sensitive and reliable assay for detecting enterotoxin genes ( nheA , entFM , hblD , cytK ) and emetic toxin ( ces ), specific primers each targeting one individual gene were designed. Propidium monoazide (PMA) was coupled with the developed multiplex PCR (mPCR) for the detection of viable B. cereus. The inclusivity and exclusivity of the PMA-mPCR was confirmed using a panel of 44 strains including 17 emetic and 9 enterotoxic B. cereus reference strains and 18 non-target strains. The limit of detection (LOD) without PMA treatment in pure DNA was 2 pg/reaction tube. The LOD of mPCR assay in pure heat-killed dead bacteria was 4.0 × 10 2 CFU/mL. Also, the LOD on the viable bacteria with or without PMA treatment was similar (3.8 × 10 2 CFU/mL) showing that the PMA treatment did not significantly decrease sensitivity. Finally, the newly developed PMA-mPCR successfully detected 4.8 × 10 3 and 3.6 × 10 3 CFU/g of viable B. cereus F4810/72 (emetic) and B. cereus ATCC 12480 (enterotoxic) reference strains, respectively, in food samples. Hence, this study combines PMA and mPCR to detect viable B. cereus with a wide range of toxin detection (5 toxins). Thus, the novel PMA-mPCR assay developed in this study is a rapid and efficient diagnostic tool for the monitoring of viable B. cereus in food samples and potentially other samples via appropriate DNA extraction. [ABSTRACT FROM AUTHOR]

Emetic and diarrheal toxins produced by endospore-formingBacillus cereuscauses a huge loss to dairy industry. Milk is the suitable medium for the growth ofB. cereus. The pathogen is of particular concern in the dairy industry because in spite of aggressive cleaning practices performed by the dairy industry, it is impossible to destroy these hydrophobic spores which adhere to the pipelines of the dairy-processing plant and further these spores may also form biofilms in the milk. The reporting rate of illness caused byB. cereusis vague, usually due to the short duration of (<24 h) the diarrheal and emetic syndromes. The study of toxins production byB. cereusisolates is essential to better determine the methods of controlling these toxins in dairy industry. [ABSTRACT FROM PUBLISHER]

Bacillus cereus is a Gram-positive endospore forming bacterium known for its wide spectrum of phenotypic traits, enabling it to occupy diverse ecological niches. Although the population structure of B. cereus is highly dynamic and rather panmictic, production of the emetic B. cereus toxin cereulide is restricted to strains with specific genotypic traits, associated with distinct environmental habitats. Cereulide is an ionophoric dodecadepsipeptide that is produced non-ribosomally by an enzyme complex with an unusual modular structure, named cereulide synthetase (Ces non-ribosomal peptide synthetase). The ces gene locus is encoded on a mega virulence plasmid related to the B. anthracis toxin plasmid pXO1. Cereulide, a highly thermo- and pH- resistant molecule, is preformed in food, evokes vomiting a few hours after ingestion, and was shown to be the direct cause of gastroenteritis symptoms; occasionally it is implicated in severe clinical manifestations including acute liver failures. Control of toxin gene expression in emetic B. cereus involves central transcriptional regulators, such as CodY and AbrB, thereby inextricably linking toxin gene expression to life cycle phases and specific conditions, such as the nutrient supply encountered in food matrices. While in recent years considerable progress has been made in the molecular and biochemical characterization of cereulide toxin synthesis, far less is known about the embedment of toxin synthesis in the life cycle of B. cereus. Information about signals acting on toxin production in the food environment is lacking. We summarize the data available on the complex regulatory network controlling cereulide toxin synthesis, discuss the role of intrinsic and extrinsic factors acting on toxin biosynthesis in emetic B. cereus and stress how unraveling these processes can lead to the development of novel effective strategies to prevent toxin synthesis in the food production and processing chain. [ABSTRACT FROM AUTHOR]

Korean fermented soybean products, such as doenjang, kochujang, ssamjang, and cho-kochujang, can harbor foodborne pathogens such as Bacillus cereus sensu lato (B. cereus sensu lato). The aim of this study was to characterize the toxin gene profiles, biochemical characteristics, and antibiotic resistance patterns of B. cereus sensu lato strains isolated from Korean fermented soybean products. Eighty-eight samples of Korean fermented soybean products purchased from retails in Seoul were tested. Thirteen of 26 doenjang samples, 13 of 23 kochujang samples, 16 of 30 ssamjang samples, and 5 of 9 cho-kochujang samples were positive for B. cereus sensu lato strains. The contamination level of all positive samples did not exceed 4 log CFU/g of food (maximum levels of Korea Food Code). Eighty-seven B. cereus sensu lato strains were isolated from 47 positive samples, and all isolates carried at least one enterotoxin gene. The detection rates of hblCDA, nheABC, cytK, and entFM enterotoxin genes among all isolates were 34.5%, 98.9%, 57.5%, and 100%, respectively. Fifteen strains (17.2%) harbored the emetic toxin gene. Most strains tested positive for salicin fermentation (62.1%), starch hydrolysis (66.7%), hemolysis (98.9%), motility test (100%), and lecithinase production (96.6%). The B. cereus sensu lato strains were highly resistant to β-lactam antibiotics such as ampicillin, penicillin, cefepime, imipenem, and oxacillin. Although B. cereus sensu lato levels in Korean fermented soybean products did not exceed the maximum levels permitted in South Korea (<104 CFU/g), these results indicate that the bacterial isolates have the potential to cause diarrheal or emetic gastrointestinal diseases. [ABSTRACT FROM AUTHOR]