Bacillus cereus NVH 0500/00 Can Adhere to Mucin but Cannot Produce Enterotoxins during Gastrointestinal Simulation

Adhesion to the intestinal epithelium could constitute an essential mechanism of Bacillus cereus pathogenesis. However, the enterocytes are protected by mucus, a secretion composed mainly of mucin glycoproteins. These may serve as nutrients and sites of adhesion for intestinal bacteria. In this study, the food poisoning bacterium B. cereus NVH 0500/00 was exposed in vitro to gastrointestinal hurdles prior to evaluation of its attachment to mucin microcosms and its ability to produce nonhemolytic enterotoxin (Nhe). The persistence of mucin-adherent B. cereus after simulated gut emptying was determined using a mucin adhesion assay. The stability of Nhe toward bile and pancreatin (intestinal components) in the presence of mucin agar was also investigated. B. cereus could grow and simultaneously adhere to mucin during in vitro ileal incubation, despite the adverse effect of prior exposure to a low pH or intestinal components. The final concentration of B. cereus in the simulated lumen at 8 h of incubation was 6.62 ± 0.87 log CFU ml⁻¹. At that point, the percentage of adhesion was approximately 6%. No enterotoxin was detected in the ileum, due to either insufficient bacterial concentrations or Nhe degradation. Nevertheless, mucin appears to retain B. cereus and to supply it to the small intestine after simulated gut emptying. Additionally, mucin may play a role in the protection of enterotoxins from degradation by intestinal components.     

Quantification methods for Bacillus cereus vegetative cells and spores in the gastrointestinal environment

There is an interest to understand the fate and behaviour of the food-borne pathogen Bacillus cereus in the gut, a challenging environment with a high bacterial background. We evaluated the current detection methods to select an appropriate strategy for B. cereus monitoring during gastrointestinal experiments. Application of quantitative real-time PCR (qPCR) in a gastrointestinal matrix required careful selection of the qPCR reaction and elaborate optimization of the DNA extraction protocol. Primer competition and depletion problems associated with qPCR reactions targeting general 16S rRNA gene can be avoided by the selection of a target sequence that is unique for and widespread among the target bacteria, such as the toxin gene nheB in the case of pathogenic B. cereus. Enumeration of B. cereus during the ileum phase was impossible by plating due to overgrowth by intestinal bacteria, while a carefully optimized qPCR enabled specific detection and quantification of B. cereus. On the other hand, plating allowed the distinction of viable, injured and dead bacteria and the germination of spores, which was not possible with qPCR. In conclusion, both plating and qPCR were necessary to yield the maximal information regarding the viability and physiology of the B. cereus population in various gastrointestinal compartments.     

Concerted Action of Sphingomyelinase and Non-Hemolytic Enterotoxin in Pathogenic Bacillus cereus

Bacillus cereus causes food poisoning and serious non-gastrointestinal-tract infections. Non-hemolytic enterotoxin (Nhe), which is present in most B. cereus strains, is considered to be one of the main virulence factors. However, a B. cereus ΔnheBC mutant strain lacking Nhe is still cytotoxic to intestinal epithelial cells. In a screen for additional cytotoxic factors using an in vitro model for polarized colon epithelial cells we identified B. cereus sphingomyelinase (SMase) as a strong inducer of epithelial cell death. Using single and double deletion mutants of sph, the gene encoding for SMase, and nheBC in B. cereus we demonstrated that SMase is an important factor for B. cereus cytotoxicity in vitro and pathogenicity in vivo. SMase substantially complemented Nhe induced cytotoxicity in vitro. In addition, SMase but not Nhe contributed significantly to the mortality rate of larvae in vivo in the insect model Galleria mellonella. Our study suggests that the role of B. cereus SMase as a secreted virulence factor for in vivo pathogenesis has been underestimated and that Nhe and SMase complement each other significantly to cause full B. cereus virulence hence disease formation.     

Rapid Characterization of Spores of Bacillus cereus Group Bacteria by Matrix-Assisted Laser Desorption-Ionization Time-of-Flight Mass Spectrometry

Matrix-assisted laser desorption-ionization (MALDI) time-of-flight mass spectrometry was used to characterize the spores of 14 microorganisms of the Bacillus cereus group. This group includes the four Bacillus species B. anthracis, B. cereus, B. mycoides, and B. thuringiensis. MALDI mass spectra obtained from whole bacterial spores showed many similarities between the species, except for B. mycoides. At the same time, unique mass spectra could be obtained for the different B. cereus and B. thuringiensis strains, allowing for differentiation at the strain level. To increase the number of detectable biomarkers in the usually peak-poor MALDI spectra of spores, the spores were treated by corona plasma discharge (CPD) or sonicated prior to MALDI analysis. Spectra of sonicated or CPD-treated spores displayed an ensemble of biomarkers common for B. cereus group bacteria. Based on the spectra available, these biomarkers differentiate B. cereus group spores from those of Bacillus subtilis and Bacillus globigii. The effect of growth medium on MALDI spectra of spores was also explored.     

Characterization of Bacillus Probiotics Available for Human Use

Bacillus species (Bacillus cereus, Bacillus clausii, Bacillus pumilus) carried in five commercial probiotic products consisting of bacterial spores were characterized for potential attributes (colonization, immunostimulation, and antimicrobial activity) that could account for their claimed probiotic properties. Three B. cereus strains were shown to persist in the mouse gastrointestinal tract for up to 18 days postadministration, demonstrating that these organisms have some ability to colonize. Spores of one B. cereus strain were extremely sensitive to simulated gastric conditions and simulated intestinal fluids. Spores of all strains were immunogenic when they were given orally to mice, but the B. pumilus strain was found to generate particularly high anti-spore immunoglobulin G titers. Spores of B. pumilus and of a laboratory strain of B. subtilis were found to induce the proinflammatory cytokine interleukin-6 in a cultured macrophage cell line, and in vivo, spores of B. pumilus and B. subtilis induced the proinflammatory cytokine tumor necrosis factor alpha and the Th1 cytokine gamma interferon. The B. pumilus strain and one B. cereus strain (B. cereus var. vietnami) were found to produce a bacteriocin-like activity against other Bacillus species. The results that provided evidence of colonization, immunostimulation, and antimicrobial activity support the hypothesis that the organisms have a potential probiotic effect. However, the three B. cereus strains were also found to produce the Hbl and Nhe enterotoxins, which makes them unsafe for human use.     

Air-Liquid Interface Biofilms of Bacillus cereus: Formation, Sporulation, and Dispersion

Biofilm formation by Bacillus cereus was assessed using 56 strains of B. cereus, including the two sequenced strains, ATCC 14579 and ATCC 10987. Biofilm production in microtiter plates was found to be strongly dependent on incubation time, temperature, and medium, as well as the strain used, with some strains showing biofilm formation within 24 h and subsequent dispersion within the next 24 h. A selection of strains was used for quantitative analysis of biofilm formation on stainless steel coupons. Thick biofilms of B. cereus developed at the air-liquid interface, while the amount of biofilm formed was much lower in submerged systems. This suggests that B. cereus biofilms may develop particularly in industrial storage and piping systems that are partly filled during operation or where residual liquid has remained after a production cycle. Moreover, depending on the strain and culture conditions, spores constituted up to 90% of the total biofilm counts. This indicates that B. cereus biofilms can act as a nidus for spore formation and subsequently can release their spores into food production environments.     

Metabolism of Bile Salts in Mice Influences Spore Germination in Clostridium difficile

Clostridium difficile, a spore-forming bacterium, causes antibiotic-associated diarrhea. In order to produce toxins and cause disease, C. difficile spores must germinate and grow out as vegetative cells in the host. Although a few compounds capable of germinating C. difficile spores in vitro have been identified, the in vivo signal(s) to which the spores respond were not previously known. Examination of intestinal and cecal extracts from untreated and antibiotic-treated mice revealed that extracts from the antibiotic-treated mice can stimulate colony formation from spores to greater levels. Treatment of these extracts with cholestyramine, a bile salt binding resin, severely decreased the ability of the extracts to stimulate colony formation from spores. This result, along with the facts that the germination factor is small, heat-stable, and water-soluble, support the idea that bile salts stimulate germination of C. difficile spores in vivo. All extracts able to stimulate high level of colony formation from spores had a higher proportion of primary to secondary bile salts than extracts that could not. In addition, cecal flora from antibiotic-treated mice was less able to modify the germinant taurocholate relative to flora from untreated mice, indicating that the population of bile salt modifying bacteria differed between the two groups. Taken together, these data suggest that an in vivo-produced compound, likely bile salts, stimulates colony formation from C. difficile spores and that levels of this compound are influenced by the commensal gastrointestinal flora.     

The Production of Bacillus cereus Enterotoxins Is Influenced by Carbohydrate and Growth Rate

Enterotoxin production is a key factor in Bacillus cereus food poisoning. Herein, the effect of the growth rate (μ) on B. cereus toxin production when grown on sucrose was studied and the Hemolytic BL enterotoxin (HBL) and nonhemolytic enterotoxin (Nhe) production by B. cereus was compared according to carbohydrate at μ = 0.2 h⁻¹. The anaerobic growth was carried out on continuous cultures in synthetic medium supplemented with glucose, fructose, sucrose, or an equimolar mixture of glucose and fructose. Concerning the HBL and Nhe enterotoxin production: (1) the highest enterotoxin production has occurred at μ = 0.2 h⁻¹ when growing on sucrose; (2) HBL production was repressed when glucose was consumed and the presence of fructose (alone or in mixture) cancelled glucose catabolite repression; (3) the consumption of sucrose increased Nhe production, which was not affected by the catabolite repression. Furthermore, analysis of the fermentative metabolism showed that whatever the μ or the carbon source, B. cereus used the mixed acid fermentation to ferment the different carbohydrates. The enterotoxin productions by this strain at μ = 0.2 h⁻¹ are highly influenced by the carbohydrates that do not involve any fermentative metabolism changes.     

Production and Characterization of Monoclonal Antibodies against the Hemolysin BL Enterotoxin Complex Produced by Bacillus cereus

A total of five hybridoma cell lines that produced monoclonal antibodies against the components of the hemolysin BL (HBL) enterotoxin complex and sphingomyelinase produced by Bacillus cereus were established and characterized. Monoclonal antibody 2A3 was specific for the B component, antibodies 1A12 and 8B12 were specific for the L₂ component, and antibody 1C2 was specific for the L₁ protein of the HBL enterotoxin complex. No cross-reactivity with other proteins produced by different strains of B. cereus was observed for monoclonal antibodies 2A3, 1A12, and 8B12, whereas antibody 1C2 cross-reacted with an uncharacterized protein of approximately 93 kDa and with a 39-kDa protein, which possibly represents one component of the nonhemolytic enterotoxin complex. Antibody 2A12 finally showed a distinct reactivity with B. cereus sphingomyelinase. The monoclonal antibodies developed in this study were also successfully applied in indirect enzyme immunoassays for the characterization of the enterotoxic activity of B. cereus strains. About 50% of the strains tested were capable of producing the HBL enterotoxin complex, and it could be demonstrated that all strains producing HBL were also highly cytotoxic.     

Development of a Streptavidin-Conjugated Single-Chain Antibody That Binds Bacillus cereus Spores

Control of microorganisms such as Bacillus cereus spores is critical to ensure the safety and a long shelf life of foods. A bifunctional single chain antibody has been developed for detection and binding of B. cereus T spores. The genes that encode B. cereus T spore single-chain antibody and streptavidin were connected for use in immunoassays and immobilization of the recombinant antibodies. A truncated streptavidin, which is smaller than but has biotin binding ability similar to that of streptavidin, was used as the affinity domain because of its high and specific affinity with biotin. The fusion protein gene was expressed in Escherichia coli BL21 (DE3) with the T7 RNA polymerase-T7 promoter expression system. Immunoblotting revealed an antigen specificity similar to that of its parent native monoclonal antibody. The single-chain antibody-streptavidin fusion protein can be used in an immunoassay of B. cereus spores by applying a biotinylated enzyme detection system. The recombinant antibodies were immobilized on biotinylated magnetic beads by taking advantage of the strong biotin-streptavidin affinity. Various liquids were artificially contaminated with 5 × 10⁴ B. cereus spores per ml. Greater than 90% of the B. cereus spores in phosphate buffer or 37% of the spores in whole milk were tightly bound and removed from the liquid phase by the immunomagnetic beads.     

A Rapid PCR-Based DNA Test for Enterotoxic Bacillus cereus

The occurrence of DNA sequences encoding the hemolysin HblA complex and Bacillus cereus enterotoxin BceT, which have recently been confirmed as enterotoxins, was studied in Bacillus spp. To amplify these DNA sequences, PCR primer systems for the B component of hblA and for bceT DNA sequences were developed. The results from the amplification of hblA sequences correlated well with results obtained with the B. cereus enterotoxin (diarrheal type) test kit (RPLA kit), but not with the results of the Bacillus diarrheal enterotoxin visual immunoassay (BDE kit). Except for two thermophilic strains, all strains that were positive in PCR amplification assays with the hblA primers were also positive when tested with the RPLA kit. The hblA DNA sequence was found in 33 strains, and these strains were closely related according to 16S rDNA-RFLP analysis, except B. pasteurii. In PCR amplifications with the bceT primers only the model strain gave a positive signal. It is concluded that screening of the hemolysin HblA complex by the PCR method allows faster detection of enterotoxin production than does testing with the RPLA enterotoxin kit.     

Bacillus cereus Spores Release Alanine that Synergizes with Inosine to Promote Germination

Background

The first step of the bacterial lifecycle is the germination of bacterial spores into their vegetative form, which requires the presence of specific nutrients. In contrast to closely related Bacillus anthracis spores, Bacillus cereus spores germinate in the presence of a single germinant, inosine, yet with a significant lag period.

Methods and Findings

We found that the initial lag period of inosine-treated germination of B. cereus spores disappeared in the presence of supernatants derived from already germinated spores. The lag period also dissipated when inosine was supplemented with the co-germinator alanine. In fact, HPLC-based analysis revealed the presence of amino acids in the supernatant of germinated B. cereus spores. The released amino acids included alanine in concentrations sufficient to promote rapid germination of inosine-treated spores. The alanine racemase inhibitor D-cycloserine enhanced germination of B. cereus spores, presumably by increasing the L-alanine concentration in the supernatant. Moreover, we found that B. cereus spores lacking the germination receptors gerI and gerQ did not germinate and release amino acids in the presence of inosine. These mutant spores, however, germinated efficiently when inosine was supplemented with alanine. Finally, removal of released amino acids in a washout experiment abrogated inosine-mediated germination of B. cereus spores.

Conclusions

We found that the single germinant inosine is able to trigger a two-tier mechanism for inosine-mediated germination of B. cereus spores: Inosine mediates the release of alanine, an essential step to complete the germination process. Therefore, B. cereus spores appear to have developed a unique quorum-sensing feedback mechanism to monitor spore density and to coordinate germination.     

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.     

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.     

Detoxification of enterotoxigenic Bacillus cereus (JX455159) isolated from meat by a local strain of Lactobacillus plantarum (JX282192)

Raw minced meat samples (25) were randomly collected from different slaughterhouses in Dakhlia and Sharkyia Governorates, Egypt. One hundred and fifty Bacillus species related to the cereus group were isolated from the collected meat samples using Mannitol Yolk Polymyxin (MYP) agar plates. Purified bacterial cultures were then tested for their virulence factors with respect to hemolysin, protease and lecithinase. Of the tested Bacillus strains (150), 81, 95.3 and 76 % of total tested Bacillus strains were positive for hemolysin, protease and lecithinase tests, respectively. The identity of one of the most potent strains suspected and encoded as Bacillus cereus F23 was confirmed by amplifying its 16S rRNA gene. The partial nucleotide sequence of the amplified 16S rRNA gene of the tested strain was submitted to GenBank with accession number JX455159. Multiplex PCR amplification of enterotoxin genes in the tested strain, using specific primers, yielded amplicons of molecular sizes 695 and 565 bp for enterotoxins hblC and cytK, respectively. Thermal resistance of B. cereus F23 (JX455159) spores was determined by calculating D values at 65, 75, 85 and 95 °C for 36, 25, 19 and 16 min, respectively, and the calculated Z value was recorded as 0.119 °C. A lactic acid bacteria (LAB) strain isolated from pickles was preliminary identified as Lactobacillus plantarum F14 (LBF14) and later confirmed by detecting its 16S rRNA gene, and it was submitted to GenBank with accession number JX282192. The identified LAB strain was tested as a bioprotective agent against toxigenic B. cereus F23 spores both in minced meat samples and BHI broth medium. A reduction in B. cereus F23 population between 4 and 6 log cycles under different tested conditions was recorded. The activity of virulence factors (protease and lecithinase) decreased and hemolytic activity was completely inhibited in the presence of 10³ CFU/ml of Lactobacillus plantarum F14 (JX282192). Inthe presence of 10⁵ CFU/ml Lactobacillus plantarum F14 (JX282192), protease and lecithinase activities of B. cereus F23 were decreased by 85 and 71 %, respectively.     

Determination of Toxigenic Potentials of <Emphasis Type="Italic">Bacillus</Emphasis> Strains Isolated from <Emphasis Type="Italic">Okpehe</Emphasis>, 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.     

Inhibition of Bacterial Growth, Enterotoxin Production, and Spore Outgrowth in Strains of Bacillus cereus by Bacteriocin AS-48

Bacteriocin AS-48 showed high bactericidal activity for mesophilic and psychrotrophic strains of Bacillus cereus over a broad pH range. AS-48 inhibition of the enterotoxin-producing strain LWL1 was enhanced by sodium nitrite, sodium lactate, and sodium chloride. The latter also enhanced AS-48 activity against strain CECT 131. Bacterial growth and enterotoxin production by strain LWL1 were completely inhibited at bacteriocin concentrations of 7.5 μg/ml. At subinhibitory bacteriocin concentrations, enterotoxin production decreased markedly and sporulation was delayed. Intact spores were resistant to AS-48 but became gradually sensitive to AS-48 during the course of germination.     

Reduction of Bacillus cereus spores in sikhye, a traditional Korean rice beverage, by modified tyndallization processes with and without carbon dioxide injection

Aims: The objective of this study was to inactivate Bacillus cereus spores in sikhye using a modified tyndallization process involving injection with carbon dioxide (CO₂). Methods and Results: Heat tolerance of B. cereus spores in tryptic soy broth and sikhye was evaluated. The D_95°C values of the B. cereus spores were 2·8–4·9 min, dependent of type of heating medium or inoculum level. The lethality of conventional heat treatment and modified tyndallization with or without CO₂ injection against B. cereus spores in sikhye was determined. The order of effectiveness was modified tyndallization with CO₂ > modified tyndallization without CO₂ > conventional heat treatment. Modified tyndallization with CO₂ reduced the number of B. cereus spores in sikhye by 5·8 log CFU ml^?1. The increased CO₂ concentration and decreased pH of sikhye resulting from CO₂ injection rapidly reverted to near‐normal values after heat treatment. Conclusions: Modified tyndallization with CO₂ was more effective than conventional heat treatment or modified tyndallization without CO₂ in reducing B. cereus spores in sikhye. Significance and Impact of the Study: Results of this study will be useful when developing strategies to control B. cereus spores in sikhye and may have application to other beverages.     

Production of Diarrheal Enterotoxins and Other Potential Virulence Factors by Veterinary Isolates of Bacillus Species Associated with Nongastrointestinal Infections

With the exceptions of Bacillus cereus and Bacillus anthracis, Bacillus species are generally perceived to be inconsequential. However, the relevance of other Bacillus species as food poisoning organisms and etiological agents in nongastrointestinal infections is being increasingly recognized. Eleven Bacillus species isolated from veterinary samples associated with severe nongastrointestinal infections were assessed for the presence and expression of diarrheagenic enterotoxins and other potential virulence factors. PCR studies revealed the presence of DNA sequences encoding hemolysin BL (HBL) enterotoxin complex and B. cereus enterotoxin T (BceT) in five B. cereus strains and in Bacillus coagulans NB11. Enterotoxin HBL was also harbored by Bacillus polymyxa NB6. After 18 h of growth in brain heart infusion broth, all seven Bacillus isolates carrying genes encoding enterotoxin HBL produced this toxin. Cell-free supernatant fluids from all 11 Bacillus isolates demonstrated cytotoxicity toward human HEp-2 cells; only one Bacillus licheniformis strain adhered to this test cell line, and none of the Bacillus isolates were invasive. This study constitutes the first demonstration that Bacillus spp. associated with serious nongastrointestinal infections in animals may harbor and express diarrheagenic enterotoxins traditionally linked to toxigenic B. cereus.