Comparison of the Tecra VIA kit, Oxoid BCET-RPLA kit and CHO cell culture assay for the detection of Bacillus cereus diarrhoeal enterotoxin

Two commercial serological kits (Oxoid BCET-RPLA and Tecra VIA) and a Chinese hamster ovary (CHO) cell cytotonicity assay for the detection of Bacillus cereus diarrhoeal enterotoxin were compared. Eleven B. cereus strains and one enterotoxigenic B. thuringiensis strain were evaluated. Both kits and the CHO cell assay yielded positive toxin responses for cell-free culture filtrates from eight out of 11 diarrhoeal enterotoxigenic strains. An emetic enterotoxin producing strain was negative with all three assays. Two B. cereus strains were negative using the BCET-RPLA kit, but positive with the Tecra VIA kit and CHO cell assay. The BCET-RPLA indicated significant levels of enterotoxin after samples were boiled, whereas the CHO cell and Tecra assays were negative. Overall, the cell culture assay was the most sensitive. However, the Tecra VIA kit provided similar results and was better suited for the rapid detection of B. cereus diarrhoeal enterotoxin.     

The hemolytic enterotoxin HBL is broadly distributed among species of the Bacillus cereus group

The prevalence of the hemolytic enterotoxin complex HBL was determined in all species of the Bacillus cereus group with the exception of Bacillus anthracis. hblA, encoding the binding subunit B, was detected by PCR and Southern analysis and was confirmed by partial sequencing of 18 strains. The sequences formed two clusters, one including B. cereus and Bacillus thuringiensis strains and the other one consisting of Bacillus mycoides, Bacillus pseudomycoides, and Bacillus weihenstephanensis strains. From eight B. thuringiensis strains, the enterotoxin gene hblA could be amplified. Seven of them also expressed the complete HBL complex as determined with specific antibodies against the L(1), L(2), and B components. Eleven of 16 B. mycoides strains, all 3 B. pseudomyoides strains, 9 of 15 B. weihenstephanensis strains, and 10 of 23 B. cereus strains carried hblA. While HBL was not expressed in the B. pseudomycoides strains, the molecular assays were in accordance with the immunological assays for the majority of the remaining strains. In summary, the hemolytic enterotoxin HBL seems to be broadly distributed among strains of the B. cereus group and relates neither to a certain species nor to a specific environment. The consequences of this finding for food safety considerations need to be evaluated.     

Detection of enterotoxic Bacillus cereus and Bacillus thuringiensis strains by PCR analysis

Many strains of Bacillus cereus cause gastrointestinal diseases, and the closely related insect pathogen B. thuringiensis has also been involved in outbreaks of diarrhea. The diarrheal types of diseases are attributed to enterotoxins. Two different enterotoxic protein complexes, hemolysin BL (HBL) and nonhemolytic enterotoxin (NHE), and an enterotoxic protein, enterotoxin T, have been characterized, and the genes have been sequenced. PCR primers for the detection of these genes were deduced and used to detect the genes in 22 B. cereus and 41 B. thuringiensis strains. At least one gene of each of the two protein complexes HBL and NHE was detected in all of the B. thuringiensis strains, while six B. cereus strains were devoid of all three HBL genes, three lacked at least two of the three NHE genes, and one lacked all three. Five different sets of primers were used for detection of the gene (bceT) encoding enterotoxin T. The results obtained with these primer sets indicate that bceT is widely distributed among B. cereus and B. thuringiensis strains and that the gene varies in sequence among different strains. PCR with the two primer sets BCET1-BCET3 and BCET1-BCET4 unambiguously detected the bceT gene, as confirmed by Southern analysis. The occurrence of the genes within the two complexes is significantly associated, while neither the occurrence of the two complexes nor the occurrence of the bceT gene is significantly associated in the 63 strains. We suggest an approach for detection of enterotoxin-encoding genes in B. cereus and B. thuringiensis based on PCR analysis with the six primer sets for the detection of genes in the HBL and NHE operons and with the BCET1, BCET3, and BCET4 primers for the detection of bceT. PCR analysis of the 16S-23S rRNA gene internal transcribed spacer region revealed identical patterns for all strains studied.     

Cloning of novel enterotoxin genes from Bacillus cereus and Bacillus thuringiensis.

A novel enterotoxin gene was cloned from Bacillus cereus FM1, and its nucleotide sequence was determined. Previously, a 45-kDa protein causing characteristic enterotoxin symptoms in higher animals had been isolated (K. Shinagawa, p. 181-193, in A. E. Pohland et al., ed., Microbial Toxins in Foods and Feeds, 1990) from the same B. cereus strain, but no report of cloning of the enterotoxin gene has been published. In the present study, a specific antibody to the purified enterotoxin was produced and used to screen the genomic library of B. cereus FM1 made with the lambda gt11 vector. An immunologically positive clone was found to contain the full protein-coding region and some 5' and 3' flanking regions. The deduced amino acid sequence of the cloned gene indicated that the protein is rich in beta structures and contains some unusual sequences, such as consecutive Asn residues. In order to clone enterotoxin genes from Bacillus thuringiensis, two PCR primers were synthesized based on the nucleotide sequence of the B. cereus gene. These primers were designed to amplify the full protein-coding region. PCR conducted with DNA preparations from the B. thuringiensis subsp. sotto and B. thuringiensis subsp. israelensis strains successfully amplified a segment of DNA with a size almost identical to that of the protein-coding region of the B. cereus enterotoxin. Nucleotide sequences of the amplified DNA segments showed that these B. thuringiensis strains contain an enterotoxin gene very similar to that of B. cereus. Further PCR screening of additional B. thuringiensis strains with four primer pairs in one reaction revealed that some additional B. thuringiensis strains contain enterotoxin-like genes.     

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.     

Specific detection of the gene for the extracellular neutral protease of Bacillus cereus by PCR and blot hybridization.

A pair of primers and a gene probe for the amplification and detection of the Bacillus cereus neutral protease gene (NPRC) were developed. Specificity for the npr genes of the B. cereus group members B. cereus, B. mycoides, and B. thuringiensis was shown. Restriction polymorphism patterns of the PCR products confirmed the presence of the NPRC gene in all three species.     

PCR assay of the groEL gene for detection and differentiation of Bacillus cereus group cells

Strains of species in the Bacillus cereus group are potentially enterotoxic. Thus, the detection of all B. cereus group strains is important. As 16S ribosomal DNA sequence analysis cannot adequately differentiate species of the B. cereus group, we explored the potential of the groEL gene as a phylogenetic marker. A phylogenetic analysis of the groEL sequences of 78 B. cereus group strains revealed that the B. cereus group strains were split into two major clusters, one including six B. mycoides and one B. pseudomycoides (cluster II) and the other including two B. mycoides and the rest of the B. cereus group strains (cluster I). Cluster I was further differentiated into two subclusters, Ia and Ib. The sodA gene sequences of representative strains from different clusters were also compared. The phylogenetic tree constructed from the sodA sequences showed substantial similarity to the tree constructed from the groEL sequences. Based on the groEL sequences, a PCR assay for detection and identification of B. cereus group strains was developed. Subsequent restriction fragment length polymorphism (RFLP) analysis verified the PCR amplicons and the differentiation of the B. cereus group strains. RFLP with MboI was identical for all the B. cereus group strains analyzed, while RFLP with MfeI or PstI classified all B. cereus and B. thuringiensis strains into two groups. All cluster II B. mycoides and B. pseudomycoides strains could be discriminated from other B. cereus group bacteria by restriction analysis with TspRI.     

Cloning and nucleotide sequence analysis of gyrB of Bacillus cereus, B. thuringiensis, B. mycoides, and B. anthracis and their application to the detection of B. cereus in rice

As 16S rRNA sequence analysis has proven inadequate for the differentiation of Bacillus cereus from closely related species, we employed the gyrase B gene (gyrB) as a molecular diagnostic marker. The gyrB genes of B. cereus JCM 2152(T), Bacillus thuringiensis IAM 12077(T), Bacillus mycoides ATCC 6462(T), and Bacillus anthracis Pasteur #2H were cloned and sequenced. Oligonucleotide PCR primer sets were designed from within gyrB sequences of the respective bacteria for the specific amplification and differentiation of B. cereus, B. thuringiensis, and B. anthracis. The results from the amplification of gyrB sequences correlated well with results obtained with the 16S rDNA-based hybridization study but not with the results of their phenotypic characterization. Some of the reference strains of both B. cereus (three serovars) and B. thuringiensis (two serovars) were not positive in PCR amplification assays with gyrB primers. However, complete sequencing of 1.2-kb gyrB fragments of these reference strains showed that these serovars had, in fact, lower homology than their originally designated species. We developed and tested a procedure for the specific detection of the target organism in boiled rice that entailed 15 h of preenrichment followed by PCR amplification of the B. cereus-specific fragment. This method enabled us to detect an initial inoculum of 0.24 CFU of B. cereus cells per g of boiled rice food homogenate without extracting DNA. However, a simple two-step filtration step is required to remove PCR inhibitory substances.     

蜡状芽胞杆菌群菌株中部分病原相关因子的检测

对26株蜡状芽胞杆菌群菌株进行了肠毒素基因及其它病原相关因子的检测。PCR结果表明,17株蜡状芽胞杆菌群菌株中含有病原调控因子plcR的同源序列。采用3组溶血肠毒素hbl基因和3组非溶血肠毒素nhe基因特异性引物,分别可从73%的菌株中至少扩增出一个与预期DNA片段大小一致的片段,其中,苏云金芽胞杆菌菌株中溶血素hbl基因和非溶血素nhe基因的阳性检出率为83%。蜡状芽胞杆菌DBt248完全没有溶血活性,而且在溶血素hbl和非溶血素nhe基因的3个亚基以及病原调控因子plcR的PCR检测中均为阴性,有望作为宿主菌用于苏云金芽胞杆菌晶体蛋白的表达和应用。     

The autolytic phenotype of the Bacillus cereus group

AIM: To determine the autolytic phenotype of five species in the Bacillus cereus group. METHODS AND RESULTS: The autolytic rate of 96 strains belonging to five species in the B. cereus group was examined under starvation conditions at pH 6, 6.5 and 8.5 in different buffers. The autolytic rate was strain-dependent with a wide variability at pH 6, but higher and more uniform at pH 6.5. At pH 8.5, and respect to the extent of autolysis at pH 6.5, it was relatively low for most of the strains with the lowest values between 13 and 52% in Bacillus mycoides and Bacillus pseudomycoides. Peptidoglycan hydrolase patterns evaluated by renaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis using cells of Bacillus thuringiensis ssp. tolworthi HD125 as an indicator, revealed complex profiles with lytic bands of about 90, 63, 46, 41, 38, 32, 28 and 25 kDa in B. cereus, B. thuringiensis and Bacillus weihenstephanensis. Bacillus mycoides and B. pseudomycoides had simpler profiles with lytic bands of 63, 46 and 38 kDa. Changes in the autolytic pattern were observed for cells harvested at the stationary phase of growth (72 h) showing an increase in the intensity of the 25 kDa band in the case of B. cereus, B. thuringiensis and B. weihenstephanensis, while no changes were observed for B. mycoides. Using Micrococcus lysodeicticus and Listeria monocytogenes as indicators lytic activity was retained by proteins of 63, 46, 38, 32 and 25 kDa and a new one of about 20 kDa in B. mycoides. Growth in the different media did not affect the autolytic pattern. NaCl abolished the activity of all the peptidoglycan hydrolases except for those of B. mycoides and B. weihenstephanensis. Lytic activity was retained in the presence of MgCl(2), MnCl(2) and EDTA and increased at basic pH. CONCLUSIONS: Bacillus cereus/B. thuringiensis/B. weihenstephanensis showed a high extent of autolysis around neutral pH, even though they presented relatively complex autolysin profiles at alkaline pH. Bacillus mycoides/B. pseudomycoides had a higher extent of autolysis at acidic pH and a simpler autolysin pattern. SIGNIFICANCE AND IMPACT OF THE STUDY: Information on the autolytic phenotype expand the phenotypic characterization of the different species in the B. cereus group.     

Detection of toxigenic strains of Bacillus cereus and other Bacillus spp. with an improved cytotoxicity assay

An improved qualitative cell cytotoxicity assay for the detection of Bacillus cereus emetic and enterotoxin is described. The presence of toxin in culture supernatant fluids was detected by measurement with the tetrazolium salt MTT, as it adversely affects the metabolic status of cultured CHO cells. Psychrotrophic B. cereus isolates (65) were assessed for toxin production using the cytotoxicity assay, and 91% of culture supernatant fluids were cytotoxic. Toxin assessment using BCET-RPLA and ELISA immunoassays indicated that 51% and 85% of the cultures, respectively, were toxigenic. There were pronounced strain differences in the amount of toxin produced by the B. cereus isolates. Some isolates of B. circulans, B. laterosporus/cereus, B. lentus, B. licheniformis, B. mycoides, B. subtilis and B. thuringiensis were also toxigenic.     

Evaluation of methods for recognising strains of the Bacillus cereus group with food poisoning potential among industrial and environmental contaminants

Toxin production, biochemical properties and ribotypes of Bacillus cereus group (B. cereus, B. thuringiensis, B. mycoides) strains originating from industrial and environmental sources (n = 64), from food poisoning incidents (n = 22) and from reference sources (n = 7) were analysed. Forty ribotypes were found among the 93 strains. Eleven strains from food poisoning incidents produced emetic (mitochondrio) toxin, as determined by the boar spermatozoa toxicity test. These strains possessed closely similar ribotypes which were rare among strains of other origins. Sperm toxin producing (cereulide positive) strains did not hydrolyse starch and did not produce haemolysin BL, as determined by the reverse passive latex agglutination test. Sixteen different ribotypes were found among B. cereus strains from board machines (n = 16) and from packaging board (n = 16), indicating many different sources of B. cereus contamination in board mills. Strains originating from packaging board had predominantly different ribotypes from those of dairy and dairy product originating strains. Nine (53%) out of 17 strains from a single dairy process shared the same ribotype whereas strains from milk and milk products from different dairies had different ribotypes indicating that B. cereus group populations were dairy specific. Twenty-two percent of strains isolated from the paperboard industry on non-selective medium were lecithinase negative, including enterotoxin producing strains. This stresses the importance of other detection methods not based on a positive lecithinase reaction.     

Differentiation of Bacillus anthracis and other 'Bacillus cereus group' bacteria using IS231-derived sequences

Abstract Sequences based on the conserved 20 bp inverted repeat of IS 231 variants were used as polymerase chain reaction-based fingerprinting primers of the member species of the Bacillus cereus group ( B. anthracis, B. cereus, B. thuringiensis and B. mycoides ), because of their close association with transposons, principally Tn 4430 in B. thuringiensis . Fingerprints of B. anthracis were simple, and specifically allowed its identification and sub-differentiation from other members of the group. Fingerprints for B. cereus were strain-specific; those for B. thuringensis gave a 1650 bp product, characteristic of 1S 231 variants A-F. The same reaction conditions gave one or two bands for both B. anthracis and B. cereus that differed by restriction endonuclease mapping from the B. thuringiensis PCR product and established IS 231 restriction maps; this does not preclude some kind of relationship between these products and IS 231 .     

Markers for species-specific detection of bacteria from Bacillus cereus group

rpoB and gyr genes (and their fragments) of chromosomal DNA of bacteria from Bacillus cereus group - B. anthracis, B. cereus, and B. thuringiensis - which are the potential markers for their genotyping were sequenced and phylogenetic trees were constructed. Sets of primers for species-specific detection of B. anthracis, B. cereus, and B. thuringiensis by multiplex polymerase chain reaction were designed. Also primers sets, which allow to differentiate strains of B. anthracis with various plasmid profiles (containing both plasmids (pXO1+, pXO2+), and without one (pXO1+, pXO2- or pXO1-, pXO2+) or both plasmids (pXO1-, pXO2-), determining pathogenic characteristics of the strains, were developed. For multiplex PCR primer sets were optimized on the annealing temperature of primers and amplicon length. Itwas shown that phylogenetic tree can be applied as an indicator of reliability and accuracy of taxonomical classification of microorganisms' species and subspecies. Comparison of pXO1 and pXO2 plasmid sequences of B. anthracis showed that these plasmids contain 18 and 4 palindrome sequences respectively which can potentially form thermodynamically stable hairpin-loop structures.     

Development of a PCR assay for identification of the Bacillus cereus group species

Aims:  A PCR technique was developed as a reliable and rapid identification method for the Bacillus cereus group species, based on a unique conserved sequence of the motB gene (encoding flagellar motor protein) from B. cereus , Bacillus thuringiensis and Bacillus anthracis .
Methods and Results:  Primer locations were identified against eight strains of the B. cereus group spp. from nucleotide sequences available in the National Centre for Biotechnology Information database. The PCR assay was applied for the identification of 117 strains of the B. cereus group spp. and 19 strains from other microbial species, with special emphasis on foodborne pathogens.
Conclusion:  The designed cross-species primers are group specific and did not react with DNA from other Bacillus and non- Bacillus species either motile or not. The primers system enabled us to detect 10³ CFU of B. cereus cells per millilitre of sample.
Significance and Impact of the Study:  Bacillus cereus group spp. belongs to one of the most prevalent foodborne pathogens. Bacterial growth results in production of different toxins; therefore, consumption of food containing >10⁶ bacteria per gram may result in emetic and diarrhoeal syndromes. A rapid and sensitive bacterial detection method is significant for food safety.     

Discrimination of Bacillus cereus and Bacillus thuringiensis with 16S rRNA and gyrB gene based PCR primers and sequencing of their annealing sites

AIMS: To evaluate the possibility for discrimination of Bacillus cereus and B. thuringiensis using 16S rRNA and gyrB gene based PCR methods, and to obtain the sequences of the primer annealing sites so that the PCR results may be explained. METHODS AND RESULTS: Based on the sequence difference in the variable region (V1) of 16S rRNA and in the gyrB gene between B. cereus and B. thuringiensis, PCR primers specific to these Bacillus spp. were designed. When these primers were used to discriminate B. cereus and B. thuringiensis, six of 82 B. cereus strains were identified as B. thuringiensis while 67 of 73 B. thuringiensis strains were identified as B. cereus. Sequence analysis of the primer annealing sites showed that there is no clear-cut difference in the V1 region of 16S rRNA, and in the gyrB gene, between B. cereus and B. thuringiensis strains. CONCLUSIONS: Although 16S rDNA based probes and gyrB gene based PCR primers have been suggested for the discrimination of B. cereus and B. thuringiensis strains, when a large number of Bacillus strains was tested, results showed that discrimination between B. cereus and B. thuringiensis is difficult. Therefore, to distinguish B. thuringiensis from B. cereus, a single feature, such as the presence of a parasporal crystal protein or cry gene, may sometimes be reliable. SIGNIFICANCE AND IMPACT OF THE STUDY: Discrimination between B. cereus and B. thuringiensis is a challenging debate to which this paper makes a contribution.     

Phenotypic and genotypic comparisons of 23 strains from the Bacillus cereus complex for a selection of known and putative B. thuringiensis virulence factors

Sixteen Bacillus thuringiensis, four Bacillus cereus and three Bacillus anthracis isolates were screened for a selection of known and putative B. thuringiensis virulence factors. PCR primers were designed to detect genes for phosphatidylcholine specific phospholipase C, phosphatidylinositol specific phospholipase C, immune inhibitor A, vegetative insecticidal protein 3A, a protein proposed to be involved in capsule synthesis, a newly identified Ser/Thr kinase homologue and enterotoxin entS. Motility, the presence of flagella, haemolysis, chitinase and lecithinase production were also evaluated. The widely varying profiles of the 23 strains from the complex provide a pool of different genotypes that can help to identify factors involved in pathogenicity.     

A comparative study of enzyme variation in Bacillus cereus and Bacillus thuringiensis

Thirty-two strains of Bacillus spp. were examined in a multilocus enzyme study by agarose gel electrophoresis. The organisms were Bacillus thuringiensis (21 strains, B. cereus (8), including two of var. mycoides, and B. megaterium (3). Strains having similar enzyme variants were grouped into zymovars. A total of 10 of 11 enzyme loci studied were polymorphic and 27 zymovars were distinguished among the 32 strains. The results were subjected to numerical analysis, phenetic affinities and genetic distances between the strains were calculated. The numerical analysis was unable to differentiate between B. thuringiensis and B. cereus. Our results indicated that based on this multilocus enzyme study these zymovars should be considered as belonging to the same species. A mycoides variant of B. cereus was the most distinctive strain studied and clearly belonged to a separate species, B. mycoides. The technique also allowed for identification of contamination and mislabelling of strains.     

A comparative study of enzyme variation in Bacillus cereus and Bacillus thuringiensis

Thirty-two strains of Bacillus spp. were examined in a multilocus enzyme study by agarose gel electrophoresis. The organisms were Bacillus thuringiensis (21 strains), B. cereus (8), including two of var. mycoides , and B. megaterium (3). Strains having similar enzyme variants were grouped into zymovars. A total of 10 of 11 enzyme loci studied were polymorphic and 27 zymovars were distinguished among the 32 strains. The results were subjected to numerical analysis, phenetic affinities and genetic distances between the strains were calculated. The numerical analysis was unable to differentiate between B. thuringiensis and B. cereus . Our results indicated that based on this multilocus enzyme study these zymovars should be considered as belonging to the same species. A mycoides variant of B. cereus was the most distinctive strain studied and clearly belonged to a separate species, B. mycoides. The technique also allowed for identification of contamination and mislabelling of strains.