Evidence for non-ribosomal peptide synthetase production of cereulide (the emetic toxin) in Bacillus cereus

Little is known about the process whereby the emetic toxin (or cereulide) of Bacillus cereus is produced. Two cereulide-producing strains of B. cereus were cloned and sequenced following polymerase chain reaction (PCR) amplification with primers that were specific for conserved regions of non-ribosomal peptide synthetase (NRPS) genes. The cloned regions of the B. cereus strains were highly homologous to conserved regions of other peptide synthetase nucleotide sequences. Primers were designed for two variable regions of the NRPS gene sequence to ensure specificity for the emetic strains. A total of 86 B. cereus strains of known emetic or non-emetic activity were screened using these primers. All of the emetic strains (n=30) displayed a 188 bp band following amplification and gel electrophoresis. We have developed an improved method of identifying emetic strains of B. cereus and provided evidence that cereulide is produced by peptide synthetases.     

Cereulide-producing strains of <Emphasis Type="Italic">Bacillus cereus</Emphasis> show diversity

Producers of cereulide, the emetic toxin of Bacillus cereus, are known to constitute a specific subset within this species. We investigated physiological and genetic properties of 24 strains of B. cereus including two high cereulide producers (600–1,800 ng cereulide mg⁻¹ wet weight biomass), seven average producers (180–600 ng cereulide mg⁻¹ wet weight biomass), four low cereulide producers (20–160 ng cereulide mg⁻¹ wet weight biomass) and 11 non-producers representing isolates from food, food poisoning, human gut and environment. The 13 cereulide producers possessed 16S rRNA gene sequences identical to each other and identical to that of B. anthracis strains Ames, Sterne from GenBank and strain NC 08234–02, but showed diversity in the adk gene (two sequence types), in ribopatterns obtained with EcoRI and PvuII (three types of patterns), in tyrosin decomposition, haemolysis and lecithin hydrolysis (two phenotypes). The cereulide-producing isolates from the human gut represented two ribopatterns of which one was novel to cereulide-producing B. cereus and two phenotypes. We conclude that the cereulide-producing B. cereus are genetically and biochemically more diverse than hitherto thought.     

A novel dodecadepsipeptide, cereulide, isolated from Bacillus cereus causes vacuole formation in HEp-2 cells

Abstract A HEp-2 cell-vacuolation factor was extracted and purified from the culture supernatant of a Bacillus cereus strain which caused emetic-syndrome food poisoning. The final preparation was chemically pure, and the toxin was named as cereulide. Mass spectrometry, NMR studies and chemical degradation revealed that the cereulide is a cyclic dodecadepsipeptide, (D-O-Leu-D-Ala- L-O-Val-L-Val)₃, which is closely related to the potassium ionophore, valinomycin.     

Synthesis of the reported structure of homocereulide and its vacuolation assay

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.     

Comparison of multiplex PCR, enzyme immunoassay and cell culture methods for the detection of enterotoxinogenic Bacillus cereus

Fast and reliable methods are needed for the detection of pathogenic Bacillus cereus which should provide consistent results. Therefore, we tested a panel of 176 strains, including B. cereus strains, B. cereus group strains and other Bacillus spp. with polymerase chain reaction, immunoassays and cytotoxicity tests and assessed the consistency of the results. A screening multiplex PCR for the detection of hbl, nhe, ces and cytK1 as well as two multiplex PCRs for the differentiation of Hbl genes (hblC, hblD, hblA) and Nhe genes (nheA, nheB, nheC) was applied. All PCRs included an internal amplification control. Component specific antibody based immunoassays were used for the detection of the three components of Hbl and Nhe and the overall cytotoxicity to Vero cells and HEp-2 cells was checked. An overall excellent correlation was obtained for the results of the three, methodically independent assays and no false-negative PCR results were seen for any of the strains tested positive in immunoassays and cytotoxicity tests. The three multiplex PCRs proved to be a facile method for the identification of enterotoxinogenic B. cereus isolates.     

Pathological effect of synthetic cereulide, an emetic toxin of Bacillus cereus, is reversible in mice

Cereulide is the causative toxin of the emetic type of food-borne illness caused by Bacillus cereus. This toxin was previously shown to be associated with fulminant liver failure in a human case. Mice were injected i.p. with synthetic cereulide and the development of histopathological changes was examined. Hepatocytes showed mitochondrial swelling with loss of cristae, and dose-dependent increase of small fatty droplets. These microsteatotic hepatocytes were distributed mainly in the pericentral area. At higher cereulide doses, massive degeneration of hepatocytes occurred. The serum values of hepatic enzymes were highest on days 2-3 after the inoculation of cereulide, and rapidly decreased thereafter. General recovery from the pathological changes and regeneration of hepatocytes was observed after 4 weeks.     

A novel dodecadepsipeptide, cereulide, is an emetic toxin of Bacillus cereus

Abstract A vacuole-formation substance, cereulide of Bacillus cereus , is an emetic toxin in animals. Both oral administration and intraperitoneal injection of cereulide caused dose-dependent emesis in Suncus murinus , a new animal model of emesis. Vagotomy or a 5-HT₃ receptor antagonist completely abolished this emetic effect. Therefore, cereulide causes emesis through the 5-HT₃ receptor and stimulation of the vagus afferent. We also found that our purified cereulide caused swelling of mitochondria of HEp-2 cells.     

Identification of emetic toxin producing Bacillus cereus strains by a novel molecular assay

Bacillus cereus causes two types of gastrointestinal diseases: emesis and diarrhea. The emetic type of the disease is attributed to the heat-stable depsipeptide cereulide and symptoms resemble Staphylococcus aureus intoxication, but there is no rapid method available to detect B. cereus strains causing this type of disease. In this study, a polymerase chain reaction (PCR) fragment of unknown function was identified, which was shown to be specific for emetic toxin producing strains of B. cereus. The sequence of this amplicon was determined and a PCR assay was developed on this basis. One hundred B. cereus isolates obtained from different food poisoning outbreaks and diverse food sources from various geographical locations and 29 strains from other species belonging to the B. cereus group were tested by this assay. In addition, 49 non-B. cereus group strains, with special emphasis on food pathogens, were used to show that the assay is specific for emetic toxin producing B. cereus strains. The presented PCR assay is the first molecular tool for the rapid detection of emetic toxin producing B. cereus strains.