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. Received: 4 December 1995 / Accepted: 30 January 1996     

Complete Genome Sequence of Bacillus cereus NC7401, Which Produces High Levels of the Emetic Toxin Cereulide

We report the complete and annotated genome sequence of Bacillus cereus NC7401, a representative of the strain group that causes emetic-type food poisoning. The emetic toxin, cereulide, is produced by a nonribosomal protein synthesis (NRPS) system that is encoded by a gene cluster on a large resident plasmid, pNCcld.     

Semiautomated Metabolic Staining Assay for Bacillus cereus Emetic Toxin

This paper describes a specific, sensitive, semiautomated, and quantitative Hep-2 cell culture-based 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay for Bacillus cereus emetic toxin. Of nine Bacillus, Brevibacillus, and Paenibacillus species assessed for emetic toxin production, only B. cereus was cytotoxic.     

Turnover of Phospholipids in Bacillus cereus under Various Growth Conditions

As a search for natural antioxidants from plant materials, strong antioxidative activity was observed in leaf waxes extracted from Eucalyptus species. A novel type of antioxidant was isolated from the leaf wax of Eucalyptus globulus and identified as n-tritriacontan-16,18-dione. Antioxidative activities were determined by different methods; a thiocyanate method, a thiobarbituric acid method, a total carbonyl value method and a weighing test. The anti-oxidant showed remarkable antioxidative activity in a water/alcohol system and was more effective than α-tocopherol and BHA; however, it has no antioxidative activity in an oil system.     

A Novel Sensitive Bioassay for Detection of Bacillus cereus Emetic Toxin and Related Depsipeptide Ionophores

Of the toxins produced by Bacillus cereus, the emetic toxin is likely the most dangerous but, due to the lack of a suitable assay, the least well known. In this paper, a new, sensitive, inexpensive, and rapid bioassay for detection of the emetic toxin of B. cereus is described. The assay is based on the loss of motility of boar spermatozoa upon 24 h of exposure to extracts of emetic B. cereus strains or contaminated food. The paralyzed spermatozoa exhibited swollen mitochondria, but no depletion of cellular ATP or damage to plasma membrane integrity was observed. Analysis of the purified toxin by electrospray tandem mass spectrometry showed that it was a dodecadepsipeptide with a mass fragmentation pattern similar to that described for cereulide. The 50% effective concentration of the purified toxin to boar spermatozoa was 0.5 ng of purified toxin ml of extended boar semen⁻¹. This amount corresponds to 10⁴ to 10⁵ CFU of B. cereus cells. No toxicity was detected for 27 other B. cereus strains up to 10⁸ CFU ml⁻¹. The detection limit for food was 3 g of rice containing 10⁶ to 10⁷ CFU of emetic B. cereus per gram. Effects similar to those provoked by emetic B. cereus toxin were also induced in boar spermatozoa by valinomycin and gramicidin at 2 and 3 ng ml of extended boar semen⁻¹, respectively. The symptoms provoked by the toxin in spermatozoa indicated that B. cereus emetic toxin was acting as a membrane channel-forming ionophore, damaging mitochondria and blocking the oxidative phosphorylation required for the motility of boar spermatozoa.     

Certhrax Toxin,an Anthrax-related ADP-ribosyltransferase from Bacillus cereus

We identified Certhrax, the first anthrax-like mART toxin from the pathogenic G9241 strain of Bacillus cereus. Certhrax shares 31% sequence identity with anthrax lethal factor from Bacillus anthracis; however, we have shown that the toxicity of Certhrax resides in the mART domain, whereas anthrax uses a metalloprotease mechanism. Like anthrax lethal factor, Certhrax was found to require protective antigen for host cell entry. This two-domain enzyme was shown to be 60-fold more toxic to mammalian cells than anthrax lethal factor. Certhrax localizes to distinct regions within mouse RAW264.7 cells by 10 min postinfection and is extranuclear in its cellular location. Substitution of catalytic residues shows that the mART function is responsible for the toxicity, and it binds NAD⁺ with high affinity (K_D = 52.3 ± 12.2 μm). We report the 2.2 Å Certhrax structure, highlighting its structural similarities and differences with anthrax lethal factor. We also determined the crystal structures of two good inhibitors (P6 (K_D = 1.7 ± 0.2 μm, K_i = 1.8 ± 0.4 μm) and PJ34 (K_D = 5.8 ± 2.6 μm, K_i = 9.6 ± 0.3 μm)) in complex with Certhrax. As with other toxins in this family, the phosphate-nicotinamide loop moves toward the NAD⁺ binding site with bound inhibitor. These results indicate that Certhrax may be important in the pathogenesis of B. cereus.     

Cereulide, the emetic toxin of Bacillus cereus, is putatively a product of nonribosomal peptide synthesis

AIMS: To determine if cereulide, the emetic toxin produced by Bacillus cereus, is produced by a nonribosomal peptide synthetase (NRPS). METHODS AND RESULTS: NC Y, an emetic strain of Bacillus cereus, was examined for a NRPS gene using PCR with primers recognizing a fragment of a NRPS gene from the cyanobacterium Microcystis. The amplicon was sequenced and compared with other gene sequences using BLAST analysis, which showed that the amplicon from strain NC Y was similar in sequence to peptide synthetase genes in other micro-organisms, including Bacillus subtilis and B. brevis, while no such sequence was found in the complete genome sequence of a nonemetic strain of B. cereus. Specific PCR primers were then designed and used to screen 40 B. cereus isolates previously implicated in outbreaks of foodborne illness. The isolates were also screened for toxin production using the MTT cell cytotoxicity assay. PCR and MTT assay screening of the B. cereus isolates revealed a high correlation between the presence of the NRPS gene and cereulide production. CONCLUSIONS: The results indicate that cereulide is produced by a NRPS complex. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study to provide evidence identifying the mechanism of production of cereulide, the emetic toxin of B. cereus. The PCR primers developed in the study allow determination of the potential for cereulide production among isolates of B. cereus.     

Quantitative analysis of cereulide, the emetic toxin of Bacillus cereus, produced under various conditions

This paper describes a quantitative and sensitive chemical assay for cereulide, the heat-stable emetic toxin produced by Bacillus cereus. The methods previously available for measuring cereulide are bioassays that give a toxicity titer, but not an accurate concentration. The dose of cereulide causing illness in humans is therefore not known, and thus safety limits for cereulide cannot be indicated. We developed a quantitative and sensitive chemical assay for cereulide based on high-performance liquid chromatography (HPLC) connected to ion trap mass spectrometry. This chemical assay and a bioassay based on boar sperm motility inhibition were calibrated with purified cereulide and with valinomycin, a structurally similar cyclic depsipeptide. The boar spermatozoan motility assay and chemical assay gave uniform results over a wide range of cereulide concentrations, ranging from 0.02 to 230 microg ml(-1). The detection limit for cereulide and valinomycin by HPLC-mass spectrometry was 10 pg per injection. The combined chemical and biological assays were used to define conditions and concentrations of cereulide formation by B. cereus strains F4810/72, NC7401, and F5881. Cereulide production commenced at the end of logarithmic growth, but was independent of sporulation. Production of cereulide was enhanced by incubation with shaking compared to static conditions. The three emetic B. cereus strains accumulated 80 to 166 microg of cereulide g(-1) (wet weight) when grown on solid medium. Strain NC7401 accumulated up to 25 microg of cereulide ml(-1) in liquid medium at room temperature (21 +/- 1 degrees C) in 1 to 3 days, during the stationary growth phase when cell density was 2 x 10(8) to 6 x 10(8) CFU ml(-1). Cereulide production at temperatures at and below 8 degrees C or at 40 degrees C was minimal.     

Lethal Toxin of Bacillus cereus I. Relationships and Nature of Toxin, Hemolysin, and Phospholipase

Bacillus cereus phospholipase was characterized as a phospholipase C by the analysis of lecithin degradation products by thin-layer and paper chromatography. Methanol in the growth menstruum inhibited completely the synthesis of phospholipase C, whereas the synthesis of lethal toxin and hemolysin were only partially inhibited. Dialysis of preformed B. cereus products against ethyl alcohol and methanol did not inactivate hemolytic, phospholipase C, or lethal activity. The hemolytic and lethal activities of culture filtrates were completely abolished by trypsin, but phospholipase C activity was resistant to inactivation. Lethal and phospholipase C properties of culture filtrates were resistant to inactivation at 45 C, whereas the hemolytic activity was completely destroyed. Lethal, hemolytic, and phospholipase C activities appeared simultaneously in a complex growth menstruum, but the kinetics of synthesis were different in all cases. Resolution of B. cereus filtrates on columns of Sephadex showed that the phospholipase C, hemolysin, and lethal toxin are distinct proteins. Evidence is also presented which suggests a correlation between the synthesis of B. cereus toxin and the period of transition from vegetative growth to sporulation. The activity of each B. cereus product was cation-independent, as opposed to cation-dependency of the phospholipase C and lethal activities of Clostridium perfringens alpha-toxin. Immunological cross-reactivity between the B. cereus products and C. perfringens alpha-toxin was not apparent; indeed, they were shown to be antigenically distinct.     

Toxin gene profiling of enterotoxic and emetic Bacillus cereus

Very different toxins are responsible for the two types of gastrointestinal diseases caused by Bacillus cereus: the diarrhoeal syndrome is linked to nonhemolytic enterotoxin NHE, hemolytic enterotoxin HBL, and cytotoxin K, whereas emesis is caused by the action of the depsipeptide toxin cereulide. The recently identified cereulide synthetase genes permitted development of a molecular assay that targets all toxins known to be involved in food poisoning in a single reaction, using only four different sets of primers. The enterotoxin genes of 49 strains, belonging to different phylogenetic branches of the B. cereus group, were partially sequenced to encompass the molecular diversity of these genes. The sequence alignments illustrated the high molecular polymorphism of B. cereus enterotoxin genes, which is necessary to consider when establishing PCR systems. Primers directed towards the enterotoxin complex genes were located in different CDSs of the corresponding operons to target two toxin genes with one single set of primers. The specificity of the assay was assessed using a panel of B. cereus strains with known toxin profiles and was successfully applied to characterize strains from food and clinical diagnostic labs as well as for the toxin gene profiling of B. cereus isolated from silo tank populations.     

Multiple-Locus Sequence Typing and Analysis of Toxin Genes in Bacillus cereus Food-Borne Isolates

In the present study we characterized 47 food-borne isolates of Bacillus cereus using multilocus sequence typing (MLST). Newly determined sequences were combined with sequences available in public data banks in order to produce the largest data set possible. Phylogenetic analysis was performed on a total of 296 strains for which MLST sequence information is available, and three main lineages—I, II, and III—within the B. cereus complex were identified. With few exceptions, all food-borne isolates were in group I. The occurrence of horizontal gene transfer (HGT) among various strains was analyzed by several statistical methods, providing evidence of widespread lateral gene transfer within B. cereus. We also investigated the occurrence of toxin-encoding genes, focusing on their evolutionary history within B. cereus. Several patterns were identified, indicating a pivotal role of HGT in the evolution of toxin-encoding genes. Our results indicate that HGT is an important element in shaping the population structure of the B. cereus complex. The results presented here also provide strong evidence of reticulate evolution within the B. cereus complex.     

Potato Crop as a Source of Emetic Bacillus cereus and Cereulide-Induced Mammalian Cell Toxicity

Bacillus cereus, aseptically isolated from potato tubers, were screened for cereulide production and for toxicity on human and other mammalian cells. The cereulide-producing isolates grew slowly, the colonies remained small (∼1 mm), tested negative for starch hydrolysis, and varied in productivity from 1 to 100 ng of cereulide mg (wet weight)⁻¹ (∼0.01 to 1 ng per 10⁵ CFU). By DNA-fingerprint analysis, the isolates matched B. cereus F5881/94, connected to human food-borne illness, but were distinct from cereulide-producing endophytes of spruce tree (Picea abies). Exposure to cell extracts (1 to 10 μg of bacterial biomass ml⁻¹) and to purified cereulide (0.4 to 7 ng ml⁻¹) from the potato isolates caused mitochondrial depolarization (loss of ΔΨm) in human peripheral blood mononuclear cells (PBMC) and keratinocytes (HaCaT), porcine spermatozoa and kidney tubular epithelial cells (PK-15), murine fibroblasts (L-929), and pancreatic insulin-producing cells (MIN-6). Cereulide (10 to 20 ng ml⁻¹) exposed pancreatic islets (MIN-6) disintegrated into small pyknotic cells, followed by necrotic death. Necrotic death in other test cells was observed only after a 2-log-higher exposure. Exposure to 30 to 60 ng of cereulide ml⁻¹ induced K⁺ translocation in intact, live PBMC, keratinocytes, and sperm cells within seconds of exposure, depleting 2 to 10% of the cellular K⁺ stores within 10 min. The ability of cereulide to transfer K⁺ ions across biological membranes may benefit the producer bacterium in K⁺-deficient environments such as extracellular spaces inside plant tissue but is a pathogenic trait when in contact with mammalian cells.     

蜡质芽孢杆菌DLSL-2发酵条件探讨及培养基优化

对蜡质芽孢杆菌(Bacilluscereus)DLSL2深层液体发酵的主要影响因子温度、转速、初始pH值等进行了单因素实验探讨,确定了最佳培养条件:温度为30℃、转速为250r/min、初始pH值为7.0。并用均匀设计法对其发酵培养基进行了优化,优化验证实验结果为7.1×109cfu/mL明显高于原发酵培养基结果3.2×109cfu/mL。     

野生Bc菌株产黑色素的研究

Bc58是一株野生蜡状芽抱杆菌菌株,经L-酪氨酸诱导后可产生红棕色色素。通过红外光谱及各种化学测定证明该色素与Sigma公司标准黑色素（Melanin）的性质相似。生测结果显示添加Bc58黑色素的Bt制剂经紫外照射5h后的LC50为16．1μg／mL,与未经紫外照射的Bt制剂的LC50 15．2μg／mL基本相同,而比未添加黑色素的Bt制剂经紫外照射后的杀虫毒力高出近1倍。经SDS—PAGE检测表明该黑色素可保护苏云金杆菌晶体蛋白在紫外光下基本不降解,表明Bc58黑色素是一种优良的紫外保护剂。     

Dynamics of boar semen motility inhibition as a semi-quantitative measurement of Bacillus cereus emetic toxin (Cereulide)

Qualitative and quantitative application of a computer assisted sperm analyzer (CASA) for detection and quantification of cereulide was described. The plot of the decrease of the percentage of boar semen progressive motility (PMOT%) in function of time and the visual inspection of curves provided a qualitative comparison between different samples (curve slope corresponds to the amount of cereulide in the sample). If the change of PMOT% over a time required for achieving PMOT% drop to 10% (DeltaPMOT%/Deltatau) is plotted against the standard curve (obtained with known concentrations of valinomycin), a semi-quantitative estimation of the amount of cereulide in the sample is obtained. An optimized CASA method was applied to determine the production of cereulide under different conditions. No cereulide was found in aerated samples and in samples incubated at 12 degrees C. The amount of cereulide produced depended on the agar medium used, type of Bacillus cereus strain and the amount of oxygen present in the atmosphere.