The Bacillus cereus bceT enterotoxin sequence reappraised

Bacillus cereus is a known opportunistic human pathogen belonging to the B. cereus group. Establishment of the pathogenesis most likely involves several gene products. One of these gene products, a single gene component named bceT, has been cloned and described from B. cereus B-4ac [Agata et al., Microbiology 141 (1995) 983-988]. However, our sequences of the bceT region from 16 B. cereus group strains showed inconsistency with the published bceT sequence. Only part of the bceT sequence had homology to our sequences. This initiated a more thorough investigation of the bceT sequence. Restriction site search and database searches intimated that the cloned bceT was created by an incidental joining of four DNA fragments during ligation. One of these fragments had 93% homology to an open reading frame (ORF 101) located within the pathogenic island of the Bacillus anthracis pXO1 virulence plasmid. We suggest that the reported enterotoxic activity of the original cloned bceT construct could be due to either the fusion gene or the fragment with homology to ORF 101 in pXO1.     

Bacteriocidal effects and inhibition of cell separation of cinnamic aldehyde on Bacillus cereus

AIMS: In this study, bacteriocidal effects of cinnamic aldehyde on Bacillus cereus were investigated. METHODS: The bacterial culture or cell suspension in 0.85% NaCl was treated with cinnamic aldehyde at a concentration of 0.3 ml l(-1). Viable cells were counted on a nutrient agar plate. Protein leakage from the cell was determined using a protein dye. Cell morphology was observed using a scanning electron microscope. RESULTS: Bacillus cereus cells were the most sensitive to cinnamic aldehyde among four different food-borne pathogens. When the cells were treated with 0.3 ml l(-1) of cinnamic aldehyde, the viable counts decreased about 6 log cycles after 6 h of incubation. The bacterial cells remained unlysed although they were killed by cinnamic aldehyde. Treatment of cinnamic aldehyde to the exponential phase cells resulted in no significant protein leakage but strong inhibition of cell separation. CONCLUSIONS: The present findings suggest that cinnamic aldehyde exhibits bacteriocidal effects and inhibition of cell separation on B. cereus. SIGNIFICANCE AND IMPACT OF THE STUDY: These data represent an interesting background for a possible mechanism for antibacterial effects of cinnamic aldehyde.     

Short communication: Bacillus cereus capable of degrading SDS shows growth with a variety of detergents

A strain of Bacillus cereus was isolated from a detergent-polluted pond. This strain showed growth with exceedingly high concentration of both anionic and non-ionic detergents. Detergent such as SDS was rapidly taken up by the cells and degraded to dodecan-1-ol by the enzyme alkylsulphatase.     

Effects of low electric current (LEC) treatment on pure bacterial cultures

AIMS: This research focused on the effects of low electric current (LEC) on the cell viability and metabolic activity of Escherichia coli and Bacillus cereus. METHODS AND RESULTS: Different LEC intensities at fixed amperage were applied, employing either graphite or copper electrode pairs, and the effects were determined by conventional cultural methods and bioindicators. On E. coli, the LEC with graphite electrodes at 5 and 10 mA led to no significant variation, but at 20 and 40 mA there was increasing inhibition of both the enzymatic activities and growth, and a reduction in ATP content. On B. cereus, similar experiments at the lower amperages did not have any inhibitor effects, however, the 40 mA current stimulated growth, ATP content and some enzymatic activities. The LEC treatment using copper electrodes caused, already at 5 mA, inhibition of bacterial growth and metabolic and enzymatic activities in both E. coli and B. cereus. CONCLUSIONS: On the basis of the obtained results using different amperages and electrodes, we can conclude that E. coli seem to be more sensitive compared with B. cereus. SIGNIFICANCE AND IMPACT OF THE STUDY: The study increases the knowledge on LEC treatment effects on the pure bacterial cultures.     

Effects of porcine bile on survival of Bacillus cereus vegetative cells and Haemolysin BL enterotoxin production in reconstituted human small intestine media

AIMS: To determine the effects of porcine bile (PB) on Bacillus cereus vegetative cells and Haemolysin BL (HBL) enterotoxin production in reconstituted small intestine media (IM). METHODS AND RESULTS: The effects of PB on the growth of B. cereus vegetative cells in reconstituted IM at PB concentrations ranging between 0 and 3.0 g l(-1) were examined. Four gastric media (GM) named GM-J broth (JB), GM-chicken, GM-milk and GM-pea were prepared by mixing equal volumes of a gastric electrolyte solution containing pepsin with JB, chicken, semi-skimmed milk and pea soup, respectively. Bacillus cereus was inoculated at approx. 2 x 10(4) CFU ml(-1) into each GM at pH 5.0 for 30 min at 37 degrees C, then mixed to the same volume of double-strength JB (IM) and PB to give concentrations of between 0 and 3.0 g of PB per litre at pH 6.5 and incubated at 37 degrees C. The diarrhoeal B. cereus strain F4430/73 grew in IM-JB, IM-chicken and IM-milk at PB concentrations of up to 0.6, 1.5 and 1.2 g l(-1), respectively. Growth was observed in IM-pea at all concentrations tested. The highest PB concentrations allowing a 3 log B. cereus increase in IM-JB, IM-chicken, IM-milk and IM-pea after a 7-10 h incubation period were 0.3, 0.9, 0.9 and 3.0 g l(-1), respectively. The effect of PB on B. cereus cells was strongest in IM-JB, followed by IM-chicken, IM-milk and IM-pea. Haemolysin BL enterotoxin was detectable in IM-chicken, IM-whole milk, IM-semi-skimmed milk and IM-pea up to PB concentrations of only 0.6, 0.6, 0.3 and 0.9 g l(-1), respectively. The diarrhoeal B. cereus strain F4433/73 behaved similarly to B. cereus strain F4430/73, whereas the food strain TZ415 was markedly more susceptible to bile. CONCLUSIONS: The tolerance of B. cereus cells to PB strongly depends on the type of food contained in the IM. Bile tolerance is also subject to strain variation. SIGNIFICANCE AND IMPACT OF THE STUDY: The probability that B. cereus cells will grow in the small intestine, produce toxins and cause diarrhoea is likely to depend on the food they are ingested with, on the bile tolerance of the B. cereus strain, and on bile concentration.     

Proteomic analysis of Bacillus cereus growing in liquid soil organic matter

Bacillus cereus is believed to be a soil bacterium, but studied solely in laboratory culture media. The aim of this study was to assess the physiology of B. cereus growing on soil organic matter by a proteomic approach. Cells were cultured to mid-exponential phase in soil extracted solubilized organic matter (SESOM), which mimics the nutrient composition of soil, and in Luria-Bertani broth as control. Silver staining of the two-dimensional gels revealed 234 proteins spots up-regulated when cells were growing in SESOM, with 201 protein spots down-regulated. Forty-three of these differentially expressed proteins were detected by Colloidal Coomassie staining and identified by matrix assisted laser desorption ionization-time of flight MS of tryptic digests. These differentially expressed proteins covered a range of functions, primarily amino acid, lipid, carbohydrate and nucleic acid metabolism. These results suggested growth on soil-associated carbohydrates, fatty acids and/or amino acids, concomitant with shifts in cellular structure.     

Development and validation of stable reference materials for food microbiology using Bacillus cereus and Clostridium perfringens spores

Aims: To develop a new type of microbiological Reference Materials (RMs), displaying long‐term stability at room temperature. The purpose was to produce and validate two batches of RMs for the enumeration of Bacillus cereus and Clostridium perfringens. Methods and Results: The RMs were based on spores of B. cereus and Cl. perfringens, adsorbed on calcium carbonate pellets. Two batches of 1000 units were manufactured and validated in compliance with ISO guide 35. After verification of their homogeneity, the stability of the ‘RM‐B. cereus’ and ‘RM‐Cl. perfringens’ batches was proven during at least 36 and 9 months, respectively, at room temperature. The validation study was completed by international collaborative trial involving 12 laboratories, allowing the validation of the assigned values. Conclusions: The methodology developed in this work enabled to produce easy‐to‐handle and cost‐effective RMs, displaying an unprecedented stability at room temperature, a good homogeneity and a precise and validated assigned value. Significance and Impact of the Study: This study revealed new paths for the development of stable microbiological RMs. Overcoming the intrinsic instability of the living cells makes it possible to produce valuable tools for the quality assurance of microbiology laboratories.     

Deletion of sigB in Bacillus cereus affects spore properties

In Bacillus cereus and other gram-positive bacteria the alternative sigma factor sigma(B) is an important regulator of the stress response. Deletion of the sigB gene generally leads to a stress-sensitive phenotype of vegetative cells. In this study, we describe the effect of the deletion of the sigB gene in B. cereus on spore properties. In particular, spores of the sigB deletion mutant showed a defect in germination upon exposure to the germinants alanine and inosine.     

Quantitative analysis of cereulide, an emetic toxin of Bacillus cereus, by using rat liver mitochondria

An emetic toxin cereulide, produced by Bacillus cereus, causes emetic food poisonings, but a method for quantitative measurement of cereulide has not been well established. A current detection method is a bioassay method using the HEp-2 cell vacuolation test, but it was unable to measure an accurate concentration. We established a quantitative assay for cereulide based on its mitochondrial respiratory uncoupling activity. The oxygen consumption in a reaction medium containing rat liver mitochondria was rapid in the presence of cereulide. Thus uncoupling effect of cereulide on mitochondrial respiration was similar to those of uncouplers 2,4-dinitrophenol (DNP), carbonylcyanide m-chlorophenylhydrazone (CCCP), and valinomycin. This method gave constant results over a wide range of cereulide concentrations, ranging from 0.05 to 100 microg/ml. The minimum cereulide concentration to detect uncoupled oxygen consumption was 50 ng/ml and increased dose-dependently to the maximum level. Semi-log relationship between the oxygen consumption rate and the cereulide concentration enables this method to quantify cereulide. The results of this method were highly reproducible as compared with the HEp-2 cell vacuolation test and were in good agreement with those of the HEp-2 cell vacuolation test. The enterotoxin of B. cereus or Staphylococcus aureus did not show any effect on the oxygen consumption, indicating this method is specific for the identification of cereulide as a causative agent of emetic food poisonings.     

Rapid genotypic detection of Bacillus anthracis and the Bacillus cereus group by multiplex real-time PCR melting curve analysis

Bacillus anthracis has four plasmid possible virulence genotypes: pXO1+/pXO2+, pXO1+/pXO2-, pXO1-/pXO2+ or pXO1-/pXO2-. Due to the lack of a specific chromosomal marker for B. anthracis, differentiation of the pXO1-/pXO2- form of B. anthracis from closely related Bacillus cereus group species is difficult. In this study, we evaluate the ability of sspE, pXO1 and pXO2 primers to discriminate individual B. anthracis and the B. cereus group genotypes using multiplex real-time PCR and melting curve analysis. Optimal conditions for successful multiplex assays have been established. Purified DNAs from 38 bacterial strains including 11 strains of B. anthracis and 18 B. cereus group strains were analyzed. Nine of the B. cereus group near-neighbor strains were shown by multilocus sequence typing to be phylogenetically proximate to the B. anthracis clade. We have demonstrated that the four plasmid genotypes of B. anthracis and B. cereus group near-neighbors were differentially and simultaneously discriminated by this assay.