A poisonous cocktail: interplay of cereulide toxin and its structural isomers in emetic Bacillus cereus.

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.

S-layer is a key element in metabolic response and entry into the stationary phase in Bacillus cereus AH187.

Bacillus cereus is a food-borne Gram-positive pathogen. The emetic reference strain B. cereus AH187 is surrounded by a proteinaceous surface layer (S-layer) that contributes to its physico-chemical surface properties, and promotes its adhesion in response to starvation conditions. The S-layer produced by B. cereus AH187 is composed of two proteins, SL2 and EA1, which are incorporated at different growth stages. Here, we showed that deletion of the genes encoding SL2 and EA1 produced viable cells, but decreased the glucose uptake rate at the start of growth, and induced extensive reorganization of the cellular and exoproteomes upon entry into the stationary phase. As a consequence, stationary cells were less resistant to abiotic stress. Taken together, our data indicate that the S-layer is crucial but comes at a metabolic cost that modulates the stationary phase response. SIGNIFICANCE: The emetic strains of Bacillus cereus are known to cause severe food poisoning, making it crucial to understand the factors contributing to their selective enrichment in foods. Most emetic strains are surrounded by a crystalline S-layer, which is a costly protein structure to produce. In this study, we used high-throughput proteomics to investigate how S-layer synthesis affects the allocation of cellular resources in the emetic B. cereus strain AH187. Our results demonstrate that the synthesis of the S-layer plays a crucial role in the pathogen's ability to thrive under stationary growth phase conditions by modulating the stress response, thereby promoting its lifestyle as an emetic pathogen. We conclude that the synthesis of the S-layer is a critical adaptation for emetic B. cereus to successfully colonize specific niches.

GC-MS analysis of organic fractions of Chrozophora tinctoria (L.) A.Juss. and their prokinetic propensity in animal models.

Chrozophora tinctoria (L.) A.Juss. is herbaceous, monecious annual plant used traditionally to cure gastrointestinal disorders. The present study was carried out to find the bioactive compounds by Gas Chromatography-Mass Spectrometry, the acetylcholinesterase inhibitory potential acute toxicity, and emetic activity present in the ethyl acetate fraction of Chrozophora tinctoria (EAFCT) and dichloromethane fraction of Chrozophora tinctoria (DCMFCT). The compounds detected in both fractions were mostly fatty acids, with about seven compounds in EAFCT and 10 in DCMFCT. These included pharmacologically active compounds such as imipramine, used to treat depression, or hexadecanoic acid methyl ester, an antioxidant, nematicide, pesticide, hypocholesterolemic, 9,12,15-Octadecatrienoic acid, ethyl ester, (Z,Z,Z)- is used as a cancer preventive, antiarthritic, antihistaminic, hepatoprotective, insectifuge, nematicide, Pentadecanoic acid, 14-methyl-, methyl ester have antifungal, antimicrobial and antioxidant activities, 10-Octadecanoic acid, methyl ester have the property to decrease blood cholesterol, Antioxidant and antimicrobial, 1-Eicosanol is used as an antibacterial, 1-Hexadecene has antibacterial, antioxidant, and antifungal activities. Both DCMFCT and EAFCT fractions inhibited acetylcholinesterase (AChE) activity with IC50 values of 10 µg and 130 µg, respectively. Both the fractions were found to be toxic in a dose-dependent manner, inducing emesis at 0.5g onward and lethargy and mortality from 3-5 g upwards. Both the fractions combined with distilled water showed highly emetic activity. The significant increase in the number of vomits was shown by EAFCT plus distilled water which are 7.50±1.29, 7.25±3.10, and 11.75±2.22 number of vomits at 1g, 2g, and 3g/kg concentration respectively, while DCMFCT plus distilled water showed 5.25±2.22, 7.50±2.52 and 10.25±2.22 number of vomits at 1g, 2, and 3g/kg correspondingly. The antiemetic standard drug metoclopramide has a higher impact against the emesis induced by both the fractions than dimenhydrinate. Metoclopramide decreases the number of vomits caused by EAFCT to 1.00±0.00, 2.00±0.00, 4.00±1.00 at 1g, 2, and 3g/kg sequentially, while dimenhydrinate decreases the number of vomits to 1.33±0.58, 2.33±1.15, 4.33±0.58 at 1g, 2, and 3g respectively. In the same way, Metochloprimide decreases the number of emesis caused by DcmCt from 5.25±2.22, 7.50±2.52, 10.25±2.22 to 1.33±0.58, 2.33±1.1, 4.33±0.58 at 1g, 2, and 3g/kg concentrations. The present study is the first documented report that scientifically validates the folkloric use of Chrozophora tinctoria as an emetic agent.

Distribution of the Emetic Toxin Cereulide in Cow Milk.

Bacillus cereus is frequently associated with food-borne intoxications, and its emetic toxin cereulide causes emesis and nausea after consumption of contaminated foods. The major source for contamination is found within contaminated raw materials containing the highly chemically resistant cereulide, independent of vegetative bacteria cells. Up to date, non-existing removal strategies for cereulide evoke the question of how the toxin is distributed within a food sample, especially cow milk. Milk samples with different milk fat contents were incubated with purified cereulide, separated by centrifugation into a lipid and an aqueous phase, and cereulide was quantified in both fractions by SIDA-LC-MS/MS. By artificially increasing the milk fat content from 0.5% to 50%, the amount of cereulide recovered in the lipid phase and could be augmented from 13.3 to 78.6%. Further, the ratio of cereulide increased in the lipid phase of milk with additional plant-based lipid (sunflower oil) to 47.8%. This demonstrated a clear affinity of cereulide towards the hydrophobic, lipid phase, aligning with cereulide's naturally strong hydrophobic properties. Therefore, an intensified cereulide analysis of lipid enriched dairy products to prevent severe cereulide intoxications or cross-contamination in processed foods is suggested.

Phenotypic homogeneity of emetic Bacillus cereus isolates in China.

Emetic Bacillus cereus strains produce a potent cereulide cytotoxin, which can cause acute and fatal cases of food poisoning. We isolated 18 emetic B. cereus strains from a food poisoning event, and from clinical and non-random food surveillance in China and phenotypic characteristics of haemolysis, starch hydrolysis, salicin fermentation, gelatin liquefaction, cytotoxicity, and susceptibility to antibiotics were assessed. All isolates were positive for haemolysis and gelatin liquefaction, and negative for starch hydrolysis and salicin fermentation. Their haemolytic potentials were intermediate to Bacillus anthracis and B. cereus ATCC 14579 (a non-emetic strain). All isolates were cytotoxic to CHO, Hep-2, and Vero cells, and were sensitive to ampicillin. The homogeneous phenotypes of emetic isolates from China are similar to the corresponding traits of European and Japanese isolates that have been characterized, suggesting highly similar phenotypes of emetic B. cereus worldwide.

Characterization and Exposure Assessment of Emetic Bacillus cereus and Cereulide Production in Food Products on the Dutch Market.

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 (>10(2) CFU/g), and 0.7% contained levels above 10(5) 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 µg/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.

Toxin Profile, Biofilm Formation, and Molecular Characterization of Emetic Toxin-Producing Bacillus cereus Group Isolates from Human Stools.

Emetic toxin-producing Bacillus cereus group species are an important problem, because the staple food for Korean is grains such as rice. In this study, we determined the prevalence (24 of 129 isolates) of emetic B. cereus in 36,745 stool samples from sporadic food-poisoning cases in Korea between 2007 and 2008. The toxin gene profile, toxin production, and biofilm-forming ability of the emetic B. cereus isolates were investigated. Repetitive element sequence polymorphism polymerase chain reaction fingerprints (rep-PCR) were also used to assess the intraspecific biodiversity of these isolates. Emetic B. cereus was present in 0.07% of the sporadic food-poisoning cases. The 24 emetic isolates identified all carried the nheABC and entFM genes and produced NHE enterotoxin. However, they did not have hemolysin BL toxin or related genes. A relationship between biofilm formation and toxin production was not observed in this study. The rep-PCR fingerprints of the B. cereus isolates were not influenced by the presence of toxin genes, or biofilm-forming ability. The rep-PCR assay discriminated emetic B. cereus isolates from nonemetic isolates, even if this assay did not perfectly discriminate these isolates. Further study on emetic isolates possessing a high degree of diversity may be necessary to evaluate the performance of the subtyping assay to discriminate emetic and nonemetic B. cereus isolates and could provide a more accurate indication of the risk from B. cereus strains.

Chemodiversity of cereulide, the emetic toxin of Bacillus cereus.

Food-borne intoxications are increasingly caused by the dodecadepsipeptide cereulide, the emetic toxin produced by Bacillus cereus. As such intoxications pose a health risk to humans, a more detailed understanding on the chemodiversity of this toxin is mandatory for the reliable risk assessment of B. cereus toxins in foods. Mass spectrometric screening now shows a series of at least 18 cereulide variants, among which the previously unknown isocereulides A-G were determined for the first time by means of UPLC-TOF MS and ion-trap MS(n) sequencing, (13)C-labeling experiments, and post-hydrolytic dipeptide and enantioselective amino acid analysis. The data demonstrate a high microheterogeneity in cereulide and show evidence for a relaxed proof reading function of the non-ribosomal cereulide peptide synthetase complex giving rise to an enhanced cereulide chemodiversity. Most intriguingly, the isocereulides were found to differ widely in their cell toxicity correlating with their ionophoric properties (e.g., purified isocereulide A showed about 8-fold higher cytotoxicity than purified cereulide in the HEp-2 assay and induced an immediate breakdown of bilayer membranes). These findings provide a substantial contribution to the knowledge-based risk assessment of B. cereus toxins in foods, representing a still unsolved challenge in the field of food intoxications.

LC-MS analysis of the emetic toxin, cereulide, produced by Bacillus cereus.

A quantitative and chemical assay of cereulide produced in the cultures by some strains of Bacillus cereus was performed on a HPLC and a ESI electrospray ion trap mass analyzer, using the synthetic cereulide as a standard. All 20 strains of emetic B. cereus were found to produce 27 - 740 ng/ml of cereulide by the LC-MS analysis. In contrast, none of the 10 diarrheal strains produced it. 10(2) cfu/ml of the cereulide producible strain with a 210 ng/ml yield was inoculated into the 10% suspensions of 14 food products, and was incubated at 32°C for 24h. The B. cereus counts in the cultures grew in the order of 10(8) to 10(9) cfu/ml, although the bacteria could not grow in fruits, and the yields of cereulide ranged from 5.18µg in curry to 0.03µg/g of raw material and/or powder material, except for fruits. These culture supernatants were also tested for the biological activity in the HEp-2 cell culture assay. Consequently, a certain correlation was shown between the yields of cereulide and the HEp-2 vacuolation activities. In addition, the supernatants were administered i.p. to 5 Suncus marinus test animals. The emetic dose was calculated to be approximately 16µg/kg.

Rapid identification of emetic Bacillus cereus by immunochromatography.

The immunochromatographic assay, which targets a marker protein co-expressed during the synthesis of cereulide by an emetic Bacillus cereus strain, was used for easily, rapidly and specifically identifying the emetic strains among B. cereus strains from various materials associated with food poisonings. All 50 of the emetic strains showed a positive reaction to the assay, but all 50 diarrheal strains had a negative reaction. The bacterial counts of 108 cfu/ml in enrichment broth and 109 cfu/ml in food-containing enrichment were required for the identification of emetic B. cereus. The present assay could identify easily and specifically the emetic type of B. cereus within 30 min by a pure culture without special techniques and instruments.

Quantification of the emetic toxin cereulide in food products by liquid chromatography-mass spectrometry using synthetic cereulide as a standard.

Bacillus cereus produces the emetic toxin cereulide, a cyclic dodecadepsipeptide that can act as a K(+) ionophore, dissipating the transmembrane potential in mitochondria of eukaryotic cells. Because pure cereulide has not been commercially available, cereulide content in food samples has been expressed in valinomycin equivalents, a highly similar cyclic potassium ionophore that is commercially available. This research tested the biological activity of synthetic cereulide and validated its use as a standard in the quantification of cereulide contents in food samples. The synthesis route consists of 10 steps that result in a high yield of synthetic cereulide that showed biological activity in the HEp-2 cell assay and the boar sperm motility assay. The activity is different in both methods, which may be attributed to differences in K(+) content of the test media used. Using cereulide or valinomycin as a standard to quantify cereulide based on liquid chromatography-mass spectrometry (LC-MS), the concentration determined with cereulide as a standard was on average 89.9% of the concentration determined using valinomycin as a standard. The recovery experiments using cereulide-spiked food products and acetonitrile as extraction solute showed that the LC-MS method with cereulide as a standard is a reliable and accurate method to quantify cereulide in food, because the recovery rate was close to 100% over a wide concentration range.

Emetic toxin producing Bacillus cereus Korean isolates contain genes encoding diarrheal-related enterotoxins.

Bacillus cereus can cause the diarrheal and emetic type of food poisoning but the symptoms of emetic food poisoning caused by B. cereus occasionally include emesis and diarrhea. The enterotoxin characteristics of emetic toxin (cereulide) producing B. cereus were needed to be determined. Therefore, forty B. cereus strains isolated from various sources in Korea were investigated for the presence of enterotoxin genes. All strains were confirmed to produce the emetic toxin using HPLC-MS methods. The rates of the nheABC, hblCDA, entFM and cytK genes amongst emetic toxin producing B. cereus strains were 82.5, 7.5, 50.0 and 27.5%, respectively. Pattern III harbored nheABC and entFM genes and pattern V processed entFM gene and were shown to be the major patterns, being present in 55.0% (21 of 40) of the emetic toxin producing B. cereus strains. Our findings revealed that 34 (85.0%) of 40 emetic toxin producing B. cereus strains isolated in Korea have the potential to cause diarrheal and emetic type of food poisoning, simultaneously. Thus, emetic toxin and enterotoxin genes should be constantly screened to provide insight into B. cereus food poisoning.

In vitro assay for human toxicity of cereulide, the emetic mitochondrial toxin produced by food poisoning Bacillus cereus.

The in vitro boar spermatozoon test was compared with the LC ion trap MS analysis for measuring the cereulide content of a pasta dish, implemented in serious emetic food poisoning caused by Bacillus cereus. Both assays showed that the poisonous food contained approximately 1.6 microg of cereulide g(-1) implying the toxic dose in human as < or =8 microg kg(-1) body weight. The threshold concentration of cereulide provoking visible mitochondrial damage in boar sperm exposed in vitro was 2 ng of cereulide ml(-1) of extended boar sperm. The same threshold value was found for cereulide extracted from the food and from the cultured bacteria. This shows that other constituents of the food did not enhance or mask the effects of cereulide. Exposure of four human cell lines (HeLa, Caco-2, Calu-3 and Paju) to cereulide showed that the threshold concentration for the loss of mitochondrial membrane potential in human cells was similar to that observed in boar sperm. Human cells and boar sperm were equally sensitive to cereulide. The results show that boar spermatozoan assay is useful for detecting cereulide concentrations toxic to humans. Spermatozoa in commercially available extended fresh boar and cryopreserved bull semen were compared, boar sperms were 100 times more sensitive to cereulide than bull sperms.

Production of an emetic toxin, cereulide, is associated with a specific class of Bacillus cereus.

The emetic toxin (cereulide) of Bacillus cereus was quantified in several isolates of B. cereus and in various food sources. When the emetic toxin was produced, vomiting-type food poisoning was observed in humans. We also found that the H-1 serovar phenotype was strongly associated with the production of cereulide and that none of the isolates that hydrolyzed starch or expressed diarrheal enterotoxin activity produced cereulide.

High-pressure liquid chromatographic analysis of sennosides A and B purgative drugs.

Procedures are described for the analysis of the main anthraquinone glycosides of senna powder, senna fruit tablets, and sennoside tablets by high-pressure liquid chromatography (HPLC). In one HPLC analysis, TLC was used to separate the glycosides prior to elution on a strong anion-exchange column with 0.1 M ammonium nitrate solution (pH 9.0) as the mobile phase. In another HPLC analysis, separation was effected using a weak anion-exchange column with 0.1 M ammonium nitrate solution (pH 5.7A) as the mobile phase.

Co-identity of the refusal and emetic principle from Fusarium-infected corn.

The structure of vomitoxin isolated from Fusarium-contaminated corn was proved to be 3,7,15-trihydroxy-12,13-epoxytrichothec-9-en-8-one. This same toxin is responsible for the "refusal phenomenon" exhibited by swine fed contaminated corn. In addition, two new substances believed to be trichothecenes were isolated from naturally infected corn. Vomitoxin was also isolated from rice inoculated with F. graminearium NRRL 5883.