Hemolysin II is more characteristic of Bacillus thuringiensis than Bacillus cereus

To investigate the distribution of the hemolysin II determinant among strains of Bacillus cereus and Bacillus thuringiensis, thirteen strains of B. cereus and fourteen strains of B. thuringiensis strains were tested for hybridization of their chromosomal DNAs with a DNA probe containing the B. cereus hemolysin II gene. In addition, the production of hemolysin II, whose activity is not inhibited by cholesterol, was tested. The presence (absence) of the hybridization response in the microorganism's genome correlated with the presence (absence) of cholesterol-unaffected hemolysin production. Only four out of thirteen B. cereus strains were found to give a positive response in hybridization experiments, whereas thirteen out of fourteen B. thuringiensis strains responded positively. DNAs from ten B. thuringiensis strains contained a 3.5 kb EcoRV fragment, which hybridized with the B. cereus hemolysin II gene probe. The 3.5 kb EcoRV DNA fragment from one of these strains (B. thuringiensis VKM-B1555) was cloned and expressed in Escherichia coli cells. The hemolysin encoded by the cloned DNA fragment was not inhibited by cholesterol and possessed all other properties of B. cereus hemolysin II. The obtained data clearly show limited distribution of hemolysin II among B. cereus strains and demonstrate that hemolysin II is more characteristic of B. thuringiensis than B. cereus.     

Production and purification of a calcium-dependent protease from Bacillus cereus BG1

The production and purification of a calcium-dependent protease by Bacillus cereus BG1 were studied. The production of the protease was found to depend specifically on the calcium concentration in the culture medium. This suggests that this metal ion is essential for the induction of protease production and/or stabilisation of the enzyme after synthesis. The calcium requirement is highly specific since other metal ions (such as Mg²⁺ and Ba²⁺, which both activate the enzyme) are not able to induce protease production. The most appropriate medium for growth and protease production comprises (g L^–1) starch 5, CaCl₂ 2, yeast extract 2, K₂HPO₄ 0.2 and KH₂PO₄ 0.2. The protease of BG1 strain was purified to homogeneity by ultrafiltration, heat treatment, gel filtration on Sephacryl S-200, ion exchange chromatography on DEAE-cellulose and, finally, a second gel filtration on Sephacryl S-200, with a 39-fold increase in specific activity and 23% recovery. The molecular weight was estimated to be 34 kDa on SDS-PAGE. The optimum temperature and pH of the purified enzyme were determined to be 60°C and 8.0, respectively, in 100 mM Tris-HCl buffer + 2 mM CaCl₂.     

Prevalence and expression of enterotoxins in Bacillus cereus and other Bacillus spp., a literature review

Members of the Bacillus genus are ubiquitous soil microorganisms and are generally considered harmless contaminants. However, a few species are known toxin producers, including the foodborne pathogen, B. cereus. This species produces two distinct types of foodborne illness, the emetic (vomit-inducing) syndrome, associated with consumption of toxin in cooked rice dishes, and the diarrheal illness seen occasionally following consumption of contaminated meats, sauces, and certain dairy products. In the latter case, illness results from the production of enterotoxins by vegetative cells in the small intestine of the host. In dairy products, the occurrence of Bacillus spp. is inevitable, and the spore-forming ability of this organism allows it to easily survive pasteurization. Many strains have been shown to grow and produce enterotoxin in dairy products at refrigeration temperatures. Evaluation of toxin gene presence and toxin expression in Bacillus spp. other than B. cereus has not been thoroughly investigated. However, the presence of natural isolates of Bacillus spp. harboring one or more enterotoxin gene(s) and subsequent demonstration of conditions which may support toxin expression holds crucial importance in the food safety arena.     

Optimization of inactivation of endospores of Bacillus cereus by antimicrobial lipopeptides from Bacillus subtilis fmbj strains using a response surface method

Bacillus subtilis fmbj can produce a lipopeptide antimicrobial substance, the main components of which are surfactin and fengycin. In this paper, the sensitivity of Bacillus cereus to antimicrobial lipopeptides from B. subtilis fmbj was observed, and the effect of the microstructure of antimicrobial lipopeptide on spores of B. cereus was investigated. At the same time, the optimization of the inactivation of antimicrobial lipopeptides to spores of B. cereus by a response surface methodology was studied. Results showed that B. cereus had high sensitivity to it, whose minimal inhibitory concentration was 156.25 μg/ml. It could result in the death of spores by destroying the structure of resting spores and sprouting spores, as was observed by transmission electron microscopy. The optimization result indicated that spores of B. cereus could be inactivated by 2 orders of magnitude when the temperature was 29.6°C, the action time was 7.6 h, and the concentration was 3.46 mg·ml⁻¹.     

Laundry detergent compatibility of the alkaline protease from Bacillus cereus

The endogenous protease activity in various commercially available laundry detergents of international companies was studied. The maximum protease activity was found at 50 degrees C in pH range 10.5-11.0 in all the tested laundry detergents. The endogenous protease activity in the tested detergents retained up to 70% on incubation at 40 degrees C for 1 h, whereas less than 30% activity was only found on incubation at 50 degrees C for 1 h. The alkaline protease from an alkalophilic strain of Bacillus cereus was studied for its compatibility in commercial detergents. The cell free fermented broth from shake flask culture of the organism showed maximum activity at pH 10.5 and 50 degrees C. The protease from B. cereus showed much higher residual activity (more than 80%) on incubation with laundry detergents at 50 degrees C for 1 h or longer. The protease enzyme from B. cereus was found to be superior over the endogenous proteases present in the tested commercial laundry detergents in comparison to the enzyme stability during the washing at higher temperature, e.g., 40-50 degrees C.     

Distribution of toxigenic Bacillus cereus in rice samples marketed in Hong Kong

Eleven of 16 samples of rice on sale in rice shops and supermarkets in Hong Kong contained Bacillus cereus. Although B. cereus counts did not exceed 100 bacteria/g in most of the positive samples, a sample of Thai red rice and a poor quality rice originating from China contained between 300 to 1000 cells/g and 10⁴ to 2×10⁵ cells/g, respectively. Nine strains produced an enterotoxin responsible for the diarrhoeal-type B. cereus food poisoning and seven of these strains also produced a haemolysin (haemolysin BL), a dermonecrotic vascular permeability factor which may be a virulence determinant in diarrhoeal illness caused by this bacterium.P.K. Lee and J.A. Buswell are with the Department of Biology, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong; K. Shinagawa is with the Department of Veterinary Medicine, Iwate University, Iwate, Japan.     

Biosorption of cadmium by a Cd2+-hyperresistant Bacillus cereus strain HQ-1 newly isolated from a lead and zinc mine

A Cd²⁺-hyperresistant bacterial strain HQ-1 was isolated from a lead–zinc mine. The strain was characterized and identified as Bacillus cereus based on morphology, physiological tests and 16S rRNA gene analysis. The minimal inhibitory concentration of Cd²⁺ for the bacterium was 0.012 mol/l. Isotherms for cadmium (Cd) biosorption by cells of B. cereus strain HQ-1 were investigated. The equilibrium data could be fitted by a Langmuir isotherm equation. The possible functional sites that might be influenced by the sorption were determined. The results indicate that this B. cereus strain has excellent potential for biosorption of Cd. Physiological characterization of the isolate also indicates possible application of this strain for bioremediation of sites with Cd contamination.     

Lethality of chlorine, chlorine dioxide, and a commercial fruit and vegetable sanitizer to vegetative cells and spores of Bacillus cereus and spores of Bacillus thuringiensis

Chlorine, chlorine dioxide (ClO₂), and a commercial raw fruit and vegetable sanitizer (Fit powder) were evaluated for their effectiveness in killing vegetative cells and spores of Bacillus cereus and spores of Bacillus thuringiensis. The ultimate goal was to use one or both species as a potential surrogate(s) for Bacillus anthracis in studies that focus on determining the efficacy of sanitizers in killing the pathogen on food contact surfaces and foods. Treatment with alkaline (pH 10.5–11.0) ClO₂ (200 mg/mL) produced by electrochemical technologies reduced populations of a five-strain mixture of vegetative cells and a five-strain mixture of spores of B. cereus by more than 5.4 and more than 6.4 log cfu/mL, respectively, within 5 min. This finding compares with respective reductions of 4.5 and 1.8 log cfu/mL resulting from treatment with 200 mg/mL chlorine. Treatment with a 1.5% acidified (pH 3.0) solution of Fit powder product was less effective, causing 2.5-log and 0.4-log cfu/mL reductions in the number of B. cereus cells and spores, respectively. Treatment with alkaline ClO₂ (85 mg/mL), acidified (pH 3.4) ClO₂ (85 mg/mL), and a mixture of ClO₂ (85 mg/mL) and Fit powder product (0.5%) (pH 3.5) caused reductions in vegetative cell/spore populations of more than 5.3/5.6, 5.3/5.7, and 5.3/6.0 log cfu/mL, respectively. Treatment of B. cereus and B. thuringiensis spores in a medium (3.4 mg/mL organic and inorganic solids) in which cells had grown and produced spores with an equal volume of alkaline (pH 12.1) ClO₂ (400 mg/mL) for 30 min reduced populations by 4.6 and 5.2 log cfu/mL, respectively, indicating high lethality in the presence of materials other than spores that would potentially react with and neutralize the sporicidal activity of ClO₂.Published by permission of the International Association for Food Protection: Journal of Food Protection (2004) 60:1702–1708This revised version was published online in April 2005 with corrections to the text and the section heading.In section Preparation of treatment solutions the phrase 22-28°C was replaced by 22±2°C.     

Acetyl-coenzyme A: arylamine N-acetyltransferases in microorganisms: screening and isolation of an enzyme from Bacillus cereus

The raw extracts of a series of microorganisms were screened for the presence of acetyl-coenzyme A: arylamine N-acetyltransferase (AAAT) using a radioactive assay with ³H-acetyl-coenzyme A and aniline as substrates. Enzyme activities were primarily detected in the soluble fractions of Bacillus and Nocardia species, and in some further soil organisms. Only strains of Bacillus cereus were able to acetylate 4-nitroaniline and 3,5-dimethyl-4-nitroaniline. The fermentation conditions for the production of the enzyme were optimized. The AAAT from one strain of Bacillus cereus was purified 24-fold and characterized.Abbreviations AAAT acetyl-coenzyme A: arylamine N-acetyltransferase - AcP acetylphosphate - CoA coenzyme A - EDTA ethylenediaminetetra-acetic acid - PTA phosphotransacetylase     

A decrease in cytotoxic and haemolytic activities by inactivation of a single enterotoxin gene in Bacillus cereus Cx5

With the ability to colonize in the guts of a broad range of mosquito larvae, Bacillus cereus Cx5 has a potential to be utilized as a new host cell for the production of mosquito-larvicidal toxins aimed for mosquito control. However, the presence of one single (entFM) and two triple (hblCDA and nheABC) enterotoxin genes were previously confirmed in strain Cx5, raising concerns in its immediate use in the environment. Cx5 cells indeed showed recognizable levels of haemolytic and Vero cell cytotoxic activities. In this study, the single enterotoxin gene in B. cereus Cx5 (entCx5) has been inactivated in order to study the relationship between the presence of this gene and the cytotoxic and haemolytic activities found in the strain. We constructed a gene disruption plasmid, pTentCx5TV2, harbouring a truncated entCx5 gene in the temperature-sensitive shuttle vector, pUCTV2. After introducing the plasmid into B. cereus Cx5, we found that the plasmid was integrated via single crossover into the chromosome as expected at the entCx5 locus, disrupting the gene. Analysis of one mutant strain revealed that Vero cell cytotoxicity and haemolytic activity of the mutant were dramatically decreased compared to that of the wild type strain. This indicates an involvement of the entCx5 gene in haemolytic and Vero cell cytotoxic activities. The results also imply that there is a high possibility to generate an effective, and safe, host cell based on B. cereus Cx5 for the production of mosquito-larvicidal toxin.     

Extending the Bacillus cereus group genomics to putative food-borne pathogens of different toxicity

The Bacillus cereus group represents sporulating soil bacteria containing pathogenic strains which may cause diarrheic or emetic food poisoning outbreaks. Multiple locus sequence typing revealed a presence in natural samples of these bacteria of about 30 clonal complexes. Application of genomic methods to this group was however biased due to the major interest for representatives closely related to Bacillus anthracis. Albeit the most important food-borne pathogens were not yet defined, existing data indicate that they are scattered all over the phylogenetic tree. The preliminary analysis of the sequences of three genomes discussed in this paper narrows down the gaps in our knowledge of the B. cereus group. The strain NVH391-98 is a rare but particularly severe food-borne pathogen. Sequencing revealed that the strain should be a representative of a novel bacterial species, for which the name Bacillus cytotoxis or Bacillus cytotoxicus is proposed. This strain has a reduced genome size compared to other B. cereus group strains. Genome analysis revealed absence of sigma B factor and the presence of genes encoding diarrheic Nhe toxin, not detected earlier. The strain B. cereus F837/76 represents a clonal complex close to that of B. anthracis. Including F837/76, three such B. cereus strains had been sequenced. Alignment of genomes suggests that B. anthracis is their common ancestor. Since such strains often emerge from clinical cases, they merit a special attention. The third strain, KBAB4, is a typical facultative psychrophile generally found in soil. Phylogenic studies show that in nature it is the most active group in terms of gene exchange. Genomic sequence revealed high presence of extra-chromosomal genetic material (about 530kb) that may account for this phenomenon. Genes coding Nhe-like toxin were found on a big plasmid in this strain. This may indicate a potential mechanism of toxicity spread from the psychrophile strain community. The results of this genomic work and ecological compartments of different strains incite to consider a necessity of creating prophylactic vaccines against bacteria closely related to NVH391-98 and F837/76. Presumably developing of such vaccines can be based on the properties of non-pathogenic strains such as KBAB4 or ATCC14579 reported here or earlier. By comparing the protein coding genes of strains being sequenced in this project to others we estimate the shared proteome, or core genome, in the B. cereus group to be 3000+/-200 genes and the total proteome, or pan-genome, to be 20-25,000 genes.     

Purification and Characterization of Protease and Chitinase from Bacillus cereus TKU006 and Conversion of Marine Wastes by These Enzymes

A chitinase- and protease-producing bacterium was isolated and identified as Bacillus cereus TKU006. The better condition on our tests for protease and chitinase production was found when the culture was shaken at 25 degrees C for 2 days in 25 mL of medium containing 2% shrimp shell powder (w/v), 0.1% K(2)HPO(4), and 0.05% MgSO(4).7H(2)O. The molecular masses of TKU006 protease and chitinase determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis were approximately 39 and 35 kDa, respectively. The optimum pH, optimum temperature, pH stability, and thermal stability of TKU006 protease and chitinase were 9, 50 degrees C, 3-11, 50 degrees C and 5, 40 degrees C, 3-11, 60 degrees C, respectively. TKU006 protease was inhibited completely by EDTA, indicating that the TKU006 protease was a metalloprotease. The TKU006 protease and chitinase retained 61%, 60%, 73%, and 100% and 60%, 60%, 71%, and 96% of its original activity in the presence of 2% Tween 20, 2% Tween 40, 2% Triton X-100, and 1 mM SDS, respectively. The antioxidant activity of TKU006 culture supernatant was determined through the scavenging ability on DPPH with 70% per milliliter. In conclusion, the novelties of the TKU006 protease and chitinase include its high stability to the surfactants and pH. Besides, with this method, we have shown that marine wastes can be utilized to generate a high-value-added product and have revealed its hidden potential in the production of functional foods.     

Purification and characterization of a solvent and detergent-stable novel protease from Bacillus cereus

A protease-producing bacterium was isolated from slaughterhouse waste samples, Hyderabad, India. It was related to Bacillus cereus on the basis of 16S rRNA gene sequencing and biochemical properties. The protease was purified to homogeneity using ammonium sulfate precipitation, and ion exchange chromatography with a fold purification of 1.8 and a recovery of 49%. The enzyme had a relative molecular weight of 28 kDa, pH and temperature optima for this protease were 10 and 60 °C. The activity was stable between a pH range of 7.0 and 12.0. The activity was inhibited by EDTA and enhanced (four-fold) by Cu²⁺ ions indicating the presence of metalloprotease. The enzyme showed extreme stability and activity even in the presence of detergents and anionic surfactants. The enzyme also showed stability in the presence of organic solvents.     

Growth Inhibitors of Lettuce Seedlings From Bacillus cereus EJ-121

The ethyl acetate extract of the Bacillus sp. EJ-121 culture broth exhibited growth inhibitory activity on a lettuce (Lactuca sativa L.) seedlings assay. Bacillus sp. EJ-121 was identified as Bacillus cereus by the morphological characteristic and nucleotide sequence of the 16S rDNA. The bioassay-guided fractionation of the ethyl acetate extract led to the isolation of two compounds. Their structures were deduced by spectroscopic methods and determined as sodium vanillate (1) and 2-aminobenzoic acid (2). Both compounds 1 and 2 inhibited more than 90% of root length at 50 ppm (0.26 and 0.36 mM, respectively) while they had a limited effect on shoot growth at the same concentration level. Roots and shoots of lettuce seedlings showed severe deterioration at 100 ppm. In order to study the fundamental structure–activity relationship, several structurally related benzoic acid derivatives were also assayed. The existence of a polar carboxyl moiety seemed to be responsible for the stronger activity.     

Detection and phylogenic analysis of one anthrax virulence plasmid pXO1 conservative open reading frame ubiquitous presented within Bacillus cereus group strains

The presence of one of the anthrax virulence plasmid pXO1 conserved fragments was analyzed in 24 Bacillus cereus and B. thuringiensis strains, including 6 B. thuringiensis subspecies, by polymerase chain reactions. Twelve out of 24 strains showed PCR-positive for an ORF101 homologous sequence. Two pXO1-ORF101-like fragments from a B. cereus B-4ac and a commercial B. thuringiensis kurstaki HD1 were cloned, sequenced and expressed in Escherichia coli. Toxicity assays revealed that the product encoded by the pXO1-ORF101-like fragment had no impact on either Vero cells or Chinese Hamster Ovary cells, suggesting that this fragment probably not contribute to enterotoxic activity. Sequence alignment of the pXO1-ORF101 from three Bacillus anthracis and ORF101-like fragments from other 12 B. cereus group isolates indicated high identity (more than 90%) and the presence of subgroup- and strain-specific SNPs among these fragments.     

Production of an alkaline protease by Bacillus cereus MCM B-326 and its application as a dehairing agent

The present investigation describes microbial production of an alkaline protease and its use in dehairing of buffalo hide. Bacillus cereus produced extracellular protease when grown on a medium containing starch, wheat bran and soya flour (SWS). The ammonium sulphate precipitated (ASP) enzyme was applied for dehairing of buffalo hide. Microscopic observation of longitudinal section of buffalo hide revealed that the epidermis was completely removed and hair was uprooted leaving empty follicles in the hide. The ASP enzyme was stable for one month at ambient temperature between 25–35 °C. Enzymatic dehairing may be a promising shift towards an environment-friendly leather processing method.     

Kinetics of plasmid transfer among <Emphasis Type="Italic">Bacillus cereus </Emphasis>group strains within lepidopteran larvae

The cry toxin encoding plasmid pHT73 was transferred from Bacillus thuringiensis subspecies kurstaki KT0 to six B. cereus group strains in three lepidopteran (Spodoptera exigua, Plutella xyllostella and Helicoverpa armigera) larvae by conjugation. The conjugation kinetics of the plasmid was precisely studied during the larval infection using a new protocol. The infections were performed with both vegetative and sporulated strains. However, larval death only occurred when infections were made with spore and toxin preparations. Likewise, spore germinations of both donor and recipient strains were only observed in killed larvae, 44–56 h post-infection. Accordingly, kinetics showed that gene transfer between B. thuringiensis strain KT0 and other B. cereus strains only took place in dead larvae among vegetatively growing bacteria. The conjugational transfer ratios varied among different strain combinations and different larvae. The highest transfer ratio reached 5.83 × 10⁻⁶ CFU/donor between the KT0 and the AW05R recipient in Helicoverpa armigera, and all transconjugants gained the ability to produce the insecticidal crystal. These results indicated that horizontal gene transfer among B. cereus group strains might play a key role for the acquisition of extra plasmids and evolution of these strains in toxin susceptible insect larvae.     

A predictive model for the growth rate of Bacillus cereus in broth by response surface methodology

A response surface methodology (RSM) was developed for predicting the growth rate of Bacillus cereus in a tryptic soy broth medium as a function of temperature (10 to 40°C), pH (5.5 to 8.5), and the NaCl concentration (0 to 8%). The primary model showed a good fit (r² = 0.920 to 0.999) to a Gompertz equation to obtain growth rates each condition. The quadratic polynomial model was found to be significant (p < 0.0001) and predicted values were found to be in good agreement with experimental values (R² value of 0.9486). The evaluation of RSM for describing the growth rate of B. cereus used the bias factor (B_f) and the accuracy factor (A_f). Both the B_f value (1.11) and the A_f value (1.50) were within acceptable ranges. This model was provided an efficient and accurate method for predicting the growth of B. cereus as a function of the controlling factors.     

Occurrence and pathogenic potential of Bacillus cereus group bacteria in a sandy loam

The major part (94%) of the Bacillus cereus-like isolates from a Danish sandy loam are psychrotolerant Bacillus weihenstephanensis according to their ability to grow at temperatures below 7 °C and/or two PCR-based methods, while the remaining 6% are B. cereus. The Bacillus mycoides-like isolates could also be␣divided into psychrotolerant and mesophilic isolates. The psychrotolerant isolates of B. mycoides could␣be discriminated from the mesophilic by the two PCR-based methods used to characterize B.␣weihenstephanensis. It is likely that the mesophilic B. mycoides strains are synonymous with Bacillus pseudomycoides, while psychrotolerant B. weihenstephanensis, like B. mycoides, are B. mycoides senso stricto. B. cereus is known to produce a number of factors, which are involved in its ability to cause gastrointestinal and somatic diseases. All the B. cereus-like and B. mycoides like isolates from the sandy loam were investigated by PCR for the presence of 12 genes encoding toxins. Genes for the enterotoxins (hemolysin BL and nonhemolytic enterotoxin) and the two of the enzymes (cereolysin AB) were present in the major part of the isolates, while genes for phospolipase C and hemolysin III were present in fewer isolates, especially among B. mycoides like isolates. Genes for cytotoxin K and the hemolysin II were only present in isolates affiliated to B. cereus. Most of the mesophilic B. mycoides isolates did not possess the genes for the nonhemolytic enterotoxin and the cereolysin AB. The presence of multiple genes coding for virulence factors in all the isolates from the B. cereus group suggests that all the isolates from the sandy loam are potential pathogens.