Cereus Enterotoxin Research Articles

Isolation and Characterization of a Psychrotolerant Toxin Producer, Bacillus weihenstephanensis, in Liquid Egg Products

A psychrotolerant bacteria of the Bacillus cereus group was found responsible for the spoilage of whole liquid egg products. By sequencing a 16S rRNA region and performing a PCR amplification of specific 16S rRNA and cspA signatures, a Bacillus weihenstephanensis was identified. Characterization of this strain shows its ability to grow in defined medium as well as in whole liquid egg at refrigerated temperatures. The strain isolated possesses genes encoding for hemolysin BL, nonhemolytic enterotoxin, and B. cereus enterotoxins and produces enterotoxins with cytotoxic activity in whole liquid egg, even at refrigerated temperatures. The isolate exhibits a clear ability to stick and form biofilms on stainless steel, the most common material used in egg breaking factories, as well as on model hydrophilic (glass) and hydrophobic (polytetrafluoroethylene) materials. These findings show the necessity to monitor for Bacillus contamination in egg products that are often used in the composition of particularly susceptible finished products such as cream, dessert, dairy, meat, and seafood.

Differentiation and Characterization by Molecular Techniques of Bacillus cereus Group Isolates from Poto Poto and Dégué, Two Traditional Cereal-Based Fermented Foods of Burkina Faso and Republic of Congo

Poto poto (a maize sourdough) and dégué (a pearl millet–based food) are two traditional African fermented foods. The molecular biology of toxigenic and pathogenic bacteria found in those foods is largely unknown. The purpose of this study was to study the phylogenetic relatedness and toxigenic potential of 26 Bacillus cereus group isolates from these traditional fermented foods. The relatedness of the isolates was evaluated with repetitive element sequence-based PCR (REP-PCR) and 16S rDNA sequencing analysis. A multiplex real-time PCR assay targeting the lef and capC genes of B. anthracis pXO1 and pXO2 plasmids and the sspE chromosomal gene of B. cereus and B. anthracis also was carried out. Melting curve analysis of the sspE amplification product was used to differentiate B. cereus from B. anthracis, and the presence of the B. cereus enterotoxin genes was determined with PCR amplification. Isolates had 15 different REP-PCR profiles, according to which they could be clustered into four groups. 16S rDNA sequencing analysis identified 23 isolates as B. cereus or B. anthracis and three isolates as B. cereus or Bacillus sp. Multiplex real-time PCR amplification indicated the absence of the lef and capC genes of B. anthracis pXO1 and pXO2 plasmids, and melting curve analysis revealed amplification of the 71-bp sspE product typical of B. cereus in all isolates instead of the 188-bp amplicon of B. anthracis, confirming the identity of these isolates as B. cereus. Four isolates had amylolytic activity. All isolates had lecithinase activity and beta-hemolytic activity. Enterotoxin production was detected in two isolates. The emetic toxin gene was not detected in any isolate. The nheB toxin gene was detected in 19 isolates by PCR amplification; one of these isolates also contained the hblD (L1) gene. The cytotoxin K cytK-1 gene was not detected, but the cytK-2 gene was clearly detected in six isolates.

Determination of Toxigenic Potentials of Bacillus Strains Isolated from Okpehe, a Nigerian Fermented Condiment

The toxigenic potential of Bacillus species isolated from the traditional fermented condiment okpehe was determined; this is aimed at selection of non-toxigenic bacilli as starter cultures to bring about production of safe product. B. subtilis and B. cereus strains isolated from okpehe were evaluated for their possible possession of virulence characteristics. Fifty isolates were screened for their ability to produce diarrhoea enterotoxin by reversed passive latex agglutination (BCET-RPLA) test kit; the result showed that 40% of the B. cereus strains were toxigenic. The ability of the selected isolates to compete in situ and in vitro toxin production during the fermentation was also determined. The enterotoxin was not detected using BCET-RPLA kit in the spontaneously fermented samples of okpehe, but the toxin was detected in the okpehe samples fermented using B. cereus enterotoxin producer in mixed starter culture fermentation. The PCR amplification of virulence genes revealed that Bacillus cereus and B. licheniformis, a strain from the B. subtilis group, contained DNA sequences encoding the haemolysin BL (hblD) enterotoxin complex. The growth ability of B. cereus strains to high population during the fermentation and the presence of detectable diarroheagenic genes in B. cereus and B. licheniformis showed that strains carrying virulence characteristics cannot be totally ruled out in traditionally fermented okpehe.

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.

Bacillus cereus may produce two or more diarrheal enterotoxins.

Bacillus cereus strains were tested for production of diarrheal enterotoxin by the reverse passive latex agglutination test and for presence of B. cereus enterotoxin gene (bceT) by polymerase chain reaction. About 50% of 56 B. cereus strains reacted positive in broth culture in the reverse passive latex agglutination test, while the bceT gene was detected in 41.1%. Sixteen percent of the strains were positive for both diarrheal enterotoxin and bceT gene. A 741 bp probe prepared from the polymerase chain reaction product detected bceT gene in all strains that were positive with the polymerase chain reaction. This study indicates a likelihood of two or more enterotoxins being produced by B. cereus which may be involved in causing diarrheal type food poisoning.

Presence of Bacillus cereus in Street Foods in Gaborone, Botswana

The purpose of this study was to evaluate the microbiological safety and quality of street foods sold in Gaborone, Botswana. A total of 148 point-of-sale composite street food samples were bought and analyzed between June 2001 and May 2002. The analysis focused on the level of contamination of various street foods with Bacillus cereus. The B. cereus (vegetative and spores), total spore, and total viable counts were determined on all the samples. Also B. cereus isolates from 444 individual point-of-sale food samples were characterized with respect to their biochemical profiles and enterotoxigenic properties. The B. cereus contamination rate for point-of-sale foods was 65%. The B. cereus counts ranged from not detectable to levels as high as 9.1 log CFU/g. Despite the high rate of contamination of some samples, generally, most samples had B. cereus counts of less than 4 log CFU/g; hence, they were of acceptable microbiological quality. Bacillus diarrheal enterotoxin was detected from 52 isolates from individual portions of meals using the B. cereus enterotoxin reversed passive latex agglutination kit. Results of the assay revealed that 59.6% of the B. cereus isolates were enterotoxigenic. Most of the enterotoxigenic isolates were obtained from vegetable samples.

Cytotoxic Potential of Industrial Strains of Bacillus sp.

The cytotoxic potential of selected strains of Bacillus licheniformis, Bacillus amyloliquefaciens, and Bacillus subtilis, used in the production of industrial enzyme products, has been assessed. Cytotoxicity was determined in Chinese hamster ovary (CHO-K1) cells by measuring total cellular metabolic activity using the tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Initially the MTT assay was validated against toxigenic strains of Bacillus cereus, to define the exact criteria for a toxigenic versus a nontoxigenic response. The assay proved sensitive to culture broths of both a diarrheagenic strain and an emetic strain of B. cereus. The enzyme-producing strains tested were nontoxic to CHO-K1 cells. Additionally it was demonstrated that our industrial strains did not react with antibodies against B. cereus enterotoxins by use of commercial antibody-based kits from Oxoid and Tecra. A short survey of the literature concerning the toxigenic potential of species within the subtilis group is included, as is a database search of known B. cereus enterotoxins against B. subtilis and B. licheniformis DNA sequences.

Pathogenic potential of fifty Bacillus weihenstephanensis strains

The aim of this study was to evaluate the food poisoning potential of strains of the new species in the Bacillus cereus group, B. weihenstephanensis. Fifty strains were tested for cytotoxicity in a Vero cell assay, and 23 of the strains were also tested for production of enterotoxin components with commercial antibody kits, and for presence of enterotoxin gene components by polymerase chain reaction (PCR). The majority of the strains (72%) were not cytotoxic, although all of the strains that were tested with PCR and commercial kits had part of at least one of the B. cereus enterotoxins Hbl, Nhe or CytK.

Pathogenic potential of fifty Bacillus weihenstephanensis strains.

The aim of this study was to evaluate the food poisoning potential of strains of the new species in the Bacillus cereus group, B. weihenstephanensis. Fifty strains were tested for cytotoxicity in a Vero cell assay, and 23 of the strains were also tested for production of enterotoxin components with commercial antibody kits, and for presence of enterotoxin gene components by polymerase chain reaction (PCR). The majority of the strains (72%) were not cytotoxic, although all of the strains that were tested with PCR and commercial kits had part of at least one of the B. cereus enterotoxins Hbl, Nhe or CytK.

Isolation and characterization of Bacillus cereus-like bacteria from faecal samples from greenhouse workers who are using Bacillus thuringiensis-based insecticides.

Since the discovery of the insecticidal activity of Bacillus thuringiensis at the beginning of the twentieth century, this bacterium has been used increasingly against various insect pests. In spite of the extensive use of B. thuringiensis, only sporadic clinical case reports have been published. In recent years, the close relationship between B. thuringiensis and the human pathogen Bacillus cereus has been confirmed. In practice, only the insecticidal activity of B. thuringiensis distinguishes the two species. However, both species are composed of thousands of isolates with varying potential for causing adverse effects in humans. The aim of this study was to employ molecular biology methods for assessment of occupational exposure to B. thuringiensis-based biopesticides by determination of specific genetic information including plasmid profiles and random amplified polymorphic DNA (RAPD). Faecal samples from 12 persons, working in Danish greenhouses, were collected for microbial analysis. Seven persons were using B. thuringiensis-based insecticides, whereas five persons were employed at greenhouses that did not use B. thuringiensis. The bacteria were isolated on B. cereus-specific solid substrate, and colonies were further identified using the polymerase chain reaction (PCR). The PCR method was used for the identification of the enterotoxin genes HblA and BceT. The expression of enterotoxins was detected with two commercial serological kits. Primers specific for 16S-23S spacer region were used to identify the bacteria as members of the B. cereus group. Several primers towards insecticidal genes have been used in order to further characterize the isolates as subspecies of B. thuringiensis. Two faecal samples from the B. thuringiensis-exposed greenhouse workers were positive for B. cereus-like bacteria. One isolate displayed intracellular crystalline inclusions characteristic of B. thuringiensis, production of and genes for B. cereus enterotoxins and it was PCR-positive for an insecticidal toxin primer set. RAPD profiles of the faecal isolate were identical to that of strains isolated from a commercial product. The methods applied have verified that the faecal isolate was identical to the B. thuringiensis isolate found in the biopesticide used. This is the first reported case of isolation of a bacterial biopesticide from human faeces. The biopesticide was shown to harbour and express enterotoxin genes. However, there is no evidence that this caused any adverse effects to the person from whom these bacteria were isolated.

Molecular epidemiology of Bacillus cereus food poisoning.

To investigate the potential use of DNA techniques in epidemiological diagnosis of Bacillus cereus food poisoning. Fifty six B. cereus isolates from milk were studied. The 56 B. cereus isolates were characterised into enterotoxin positive (27 isolates) and enterotoxin negative (29 isolates) using reverse passive latex agglutination technique. Plasmid and genomic DNA were isolated from all the B. cereus isolates. The plasmid DNA was analysed by gel electrophoresis, while genomic DNA was used for restriction endonuclease and toxin gene analyses. Plasmid profile analysis, restriction endonuclease analysis of genomic DNA, and test for bceT and hblA genes by polymerase chain reaction and gene probing. Seventy two per cent of the isolates contained one to five plasmids of molecular sizes between 0.1 to 60 mDa. Restriction analysis of genomic DNA gave different restriction patterns among enterotoxin positive and enterotoxin negative isolates. A polymerase chain reaction assay detected bceT gene in 41.1% of the isolates, 16% of which tested positive for enterotoxin with B. cereus enterotoxin reverse passive latex agglutination (BCET-RPLA) kit, while hblA gene was detected in all the enterotoxin positive isolates. BceT and hblA gene probes detected the respective genes in all the isolates that also tested positive for toxin genes by polymerase chain reaction. DNA techniques provide an alternative approach to the diagnosis of enterotoxigenic B. cereus.

Putative virulence factor expression by clinical and food isolates of Bacillus spp. after growth in reconstituted infant milk formulae.

Forty-seven strains representing 14 different Bacillus species isolated from clinical and food samples were grown in reconstituted infant milk formulae (IMF) and subsequently assessed for adherence to, invasion of, and cytotoxicity toward HEp-2 and Caco-2 cells. Cell-free supernatant fluids from 38 strains (81%) were shown to be cytotoxic, 43 strains (91%) adhered to the test cell lines, and 23 strains (49%) demonstrated various levels of invasion. Of the 21 Bacillus cereus strains examined, 5 (24%) were invasive. A larger percentage of clinically derived Bacillus species (20%) than of similar species tested from the food environment were invasive. Increased invasion occurred after growth of selected Bacillus species in reconstituted IMF containing glucose. While PCR primer studies revealed that many different Bacillus species contained DNA sequences encoding the hemolysin BL (HBL) enterotoxin complex and B. cereus enterotoxin T, not all of these isolates expressed these diarrheagenic genes after growth in reconstituted IMF. Of the 47 Bacillus isolates examined, 3 isolates of B. cereus and 1 isolate of B. subtilis produced the HBL enterotoxin after 18 h of growth in brain heart infusion broth. However, eight isolates belonging to the species B. cereus, B. licheniformis, B. circulans, and B. megaterium were found to produce this enterotoxin after growth in reconstituted IMF when assessed with the B. cereus enterotoxin (diarrheal type) reversed passive latex agglutination (RPLA) kit. It is concluded that several Bacillus species occurring occasionally in clinical specimens and food samples are of potential medical significance due to the expression of putative virulence factors.

Bacillus cereus Group Strains, Their Hemolysin BL Activity, and Their Detection in Foods Using a 16S RNA and Hemolysin BL Gene-Targeted Multiplex Polymerase Chain Reaction System

Hemolysin BL (HBL) is a major virulence factor for Bacillus cereus group strains. It is also a target enterotoxin for the most commonly used B. cereus detection kit, i.e., the B. cereus enterotoxin (diarrheal type) reversed passive latex agglutination (BCET-RPLA) test kit. A survey of the HBL activities and the cytotoxicities to the Chinese hamster ovary (CHO) cells for the B. cereus group strains, however, showed that although only part of the B. cereus group strains are HBL active, all strains show cytotoxicity to the CHO cells. Thus, methods that allow the detection of not only the HBL but also of the B. cereus group strains are important. In this study, by comparison of the gene sequences of the 16S rRNA for B. cereus group and other bacteria strains, we designed primers B16S1 and B16S2 specific to all the B. cereus group strains. In addition, because HBL is a major enterotoxin, we also designed HBL gene-specific polymerase chain reaction (PCR) primers, i.e., Hm1 and Hm2, that generated the same results as those of the hemolysis and BCET-RPLA assays. Primers B16S1/B16S2 and Hm1/Hm2 could be combined into a multiplex PCR system for the simultaneous detection of B. cereus group cells and the possible presence of their HBL enterotoxins. Also, all these PCR systems allowed the detection of n × 100 CFU B. cereus cells per g of food sample if an 8-h enrichment step was performed prior to the PCR.

A new cytotoxin from Bacillus cereus that may cause necrotic enteritis.

A cytotoxin (CytK) has been isolated from a Bacillus cereus strain that caused a severe food poisoning outbreak killing three people. A protein of 34 kDa was highly cytotoxic, and the addition of other secreted proteins gave no synergistic effect. CytK was also necrotic and haemolytic. No known B. cereus enterotoxins were produced by this strain. A DNA sequence from 1.8 kb upstream to 0.2 kb downstream of the toxin gene was sequenced. The deduced amino acid sequence of the toxin showed similarity to Staphylococcus aureus leucocidins, gamma-haemolysin and alpha-haemolysin, Clostridium perfringens beta-toxin and B. cereus haemolysin II, all belonging to a family of beta-barrel channel-forming toxins. There was no sequence similarity between CytK and enterotoxins of B. cereus. The upstream sequence contained a partial sequence of a putative histidine kinase gene. A recognition site for PlcR, which regulates the transcription of enterotoxins HBL and Nhe of B. cereus, was found in the promoter region of the toxin. This new cytotoxin may be responsible for a disease that is similar to, although not as severe as, the necrotic enteritis caused by the beta-toxin of C. perfringens type C.

Enterotoxigenic profiles and polymerase chain reaction detection of Bacillus cereus group cells and B. cereus strains from foods and food-borne outbreaks.

Bacillus cereus is one of the important food pathogens. Since B. cereus group cells, such as B. cereus, B. thuringiensis, B. anthracis and B. mycoides, share many phenotypical properties and a high level of chromosomal sequence similarity, it is interesting to investigate the virulence profiles for B. cereus group cells, including B. cereus strains isolated from foods and samples associated with food-poisoning outbreaks. For this investigation, the presence of enterotoxin genes, such as those of haemolysin BL, B. cereus enterotoxin T and enterotoxin FM, were assayed by polymerase chain reaction (PCR) methods. Meanwhile, their enterotoxin activities were assayed using the BCET-RPLA kit, haemolytic patterns on sheep blood agar and their cytotoxicity to Chinese hamster ovary (CHO) cells. Results showed that there were 12 enterotoxigenic profiles for the 98 B. cereus group strains collected. In addition, if any of the three types of enterotoxins was present in the B. cereus group cells, these cells were shown to be cytotoxic to the CHO cells. Similar enterotoxigenic profiles could be found among strains of B. cereus, B. mycoides and B. thuringiensis. Thus, all B. cereus group strains may be potentially toxigenic and the detection of these cells in foods is important. We thus designed PCR primers, termed Ph1/Ph2, from the sphingomyelinase gene of B. cereus cells. These primers were specific for all B. cereus group strains and could be used for the detection of B. cereus cells contaminated in food samples.

Bacillus cereus may produce two or more diarrheal enterotoxins

Bacillus cereus strains were tested for production of diarrheal enterotoxin by the reverse passive latex agglutination test and for presence of B. cereus enterotoxin gene ( bceT) by polymerase chain reaction. About 50% of 56 B. cereus strains reacted positive in broth culture in the reverse passive latex agglutination test, while the bceT gene was detected in 41.1%. Sixteen percent of the strains were positive for both diarrheal enterotoxin and bceT gene. A 741 bp probe prepared from the polymerase chain reaction product detected bceT gene in all strains that were positive with the polymerase chain reaction. This study indicates a likelihood of two or more enterotoxins being produced by B. cereus which may be involved in causing diarrheal type food poisoning.

The bacteriological quality of hospital-prepared infant feeds

Twenty-four pasteurized infant feeds, prepared in a Glasgow hospital, were examined microbiologically. All produced a satisfactory total aerobic mesophilic count of ≤ 1·0 × 10 4 cfu/g (mean 6·3 × 10 1 cfu/g) within 1 h of preparation. Bacillus cereus was detected in two infant feeds immediately after preparation and one of these had a B. cereus count of 1·4 × 10 3 cfu/g exceeding the recommended safety limit of ≤ 1·0 × 10 3 cfu/g. Subsequent storage over a 14 h period at 25 °C or greater resulted in the appearance of B. cereus in a further eight feeds, the majority of which exceeded the safety limit of 10 3 cfu/g. The microbiological quality of each infant feed depended on the type and number of organisms initially present, and on the temperature and duration of storage. Incubation of feeds at ≤ 10 °C for 14 h did not alter the microbiological quality ( P = 0·05). While Bacillus licheniformis and Bacillus subtilis were the predominant organisms isolated within 8 h of incubation (45·8 and 20·8% of feeds, respectively), additional storage resulted in the emergence of B.cereus I (25%) and II (20·8%) as dominant Bacillus spp. The addition of glucose polymers and other supplements to infant formulae did not affect the type and number of organisms present ( P = 0·05). Diarrhoeal enterotoxin was detected in three of the five formulations which supported the growth of B. cereus II via the B. cereus enterotoxin reverse phase latex agglutination test BCET-RPLA system. Although the infant feeds were of similar microbiological quality ( P = 0·05), the majority of Bacillus spp. isolated have been previously implicated in either foodborne illnesses and/or opportunist infections.

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.

Evaluation of the Oxoid BCET-RPLA Kit for the Detection of Bacillus cereus Diarrheal Enterotoxin as Compared to Cell Culture Cytotonicity

The relationship between detection of heat-labile enterotoxin production by Bacillus cereus using the Oxoid BCET-RPLA kit and the production of a cytotonic response in HEp-2 and CHO cells was evaluated using 12 isolates. The strains were cultured in brain heart infusion + 0.1% glucose for 5 h at 37°C on a rotary shaker. Cell-free supernatants were prepared by filtration and tested using the two cell lines and the immunoassay. The primary biological response noted with the cell lines was cytotonicity, with the CHO cells being consistently more sensitive than the HEp-2 cells. While there was general agreement between the cell culture tests and the immunoassay, differences were noted for specific strains. Further, while activity in cell cultures was lost when cell-free supernatants were heated to 80°C for 5 min, the BCET-RPLA kits continued to detect significant levels of enterotoxin. The results suggest that confirmation of B. cereus enterotoxin using a suitable biological response may be warranted until additional research can clarify the specificity of the BCET-RPLA assay.

Development of a fluorescent immunodot assay for Bacillus cereus enterotoxin

A fluorescent immunodot assay has been developed for rapid, specific detection of B. cereus enterotoxin. None of the other Bacillus species tested showed cross-reactivity in the assay with antiserum to purified B. cereus enterotoxin. The assay can detect ≥ 50 ng of purified enterotoxin. Using this assay system, enterotoxin was found to be produced by 25 of 25 foodborne disease-related isolates and 22 of 25 isolates not related to foodborne disease (isolates from routine surveillance foods). Because of the apparent widespread ability of isolates to produce enterotoxin the assay may have potential as a rapid identification procedure for B. cereus. The substrate gel system described may have wider application in other immunoassay systems using a membrane solid phase.