Diarrheal Food Poisoning Research Articles

Effects of camellia saponins on biofilm formation and virulence factor genes of Bacillus cereus

Bacillus cereus is a common foodborne pathogen that can cause vomiting or diarrheal food poisoning and may not be suppressed using conventional cleaning procedures due to its ability to form biofilms. Biofilms have brought enormous challenges to the food safety industry. Camellia saponins are a class of oleanolanetype pentacyclic triterpenoid saponins that naturally exist in Camellia oleifera. This study demonstrated that the MBIC of camellia saponins against B. cereus was 64 mg/mL, and that the inhibitory effects on B. cereus upon adding saponins at different time periods varied as shown via scanning electron microscopy. However, the best inhibitory effect on biofilms was achieved through the addition of 1 mL of camellia saponins (1/2 MBIC) initially and adding 1 mL of camellia saponins (1/2 MBIC) again after 12 h, resulting in a decreased secretion of extracellular polymers and weakening the ability of the bacteria to form biofilms. The results showed that the swarming motility was significantly inhibited by camellia saponins. Furthermore, in the process of biofilm formation, camellia saponins could inhibit the production of extracellular proteins. qRT-PCR showed that the expression of six enterotoxin B. cereus virulence genes (hblA, hblD, cerA, cerB, nheA and hlyIII) was significantly inhibited by camellia saponins.

Dual-wavelength ratiometric immunosensor for Bacillus cereus: Oxidase-like MnO2-Au trigged “OFF-ON” detection strategy

As an opportunistic pathogen, Bacillus cereus (B. cereus) has caused increasing emetic and diarrheal food poisoning, even fulminant hepatic failure and death case. Herein, a mimetic oxidase-mediated ratiometric fluorescence enzyme-linked immunosorbent assay (ELISA) was developed to detect B. cereus. In this system, 3D MnO2-Au nanoflowers with layered crystalline structure can oxidize o-phenylenediamine (OPD) to generate a fluorescence peak at 570 nm (I570, response signal), while blue carbon quantum dots (bCDs) display a fluorescence peak at 455 nm (I455, reference signal). Under the effect of fluorescence resonance energy transfer (FRET), the response signal increased while the reference signal was weakened. By using MnO2-Au as the mimetic oxidase to replace the horseradish peroxidase and associate with antibody in traditional ELISA, a linear relationship was established between the ratiometric fluorescence signal (I570/I455) and the logarithm of B. cereus concentrations in the range from 1.7 × 102 to 3.3 × 106 CFU/mL. Comparing with conventional sandwich ELISA, the detection time was shortened by 43 min and the sensitivity was improved by 5-folds, indicating that the proposed ratiometric immunoassay holds great potential as a rapid and convenient tool for sensitive screening of B. cereus.

A novel method for direct detection of Bacillus cereus toxin genes in powdered dairy products

Bacillus cereus is a major foodborne pathogen with the potential of causing emetic and diarrhoeal food poisoning. This study reports on the development of a sensitive and reliable method that involves direct DNA extraction from powdered milk and infant formula and a multiplex PCR for detection of four B. cereus toxin genes (nheA, hblD, cytK2 and cesB). Detection limits were 2 × 102 cfu g−1 for enterotoxin genes and 2 × 103 cfu g−1 for both the enterotoxin and emetic genes. Determination of B. cereus toxin genes in powdered dairy food samples revealed five toxigenic groups. Groups I to IV contained enterotoxin genes while group V, enterotoxin and emetic genes. The results indicate that the molecular method developed is a rapid and efficient diagnostic tool for direct detection and monitoring of B. cereus toxin genes in powdered milk and infant formula and possibly in other powdered foods.

Rapid detection of viable Bacillus cereus emetic and enterotoxic strains in food by coupling propidium monoazide and multiplex PCR (PMA-mPCR)

Bacillus cereus can cause emetic and diarrheal food poisoning. It is widespread in nature and therefore, considered a major foodborne pathogen. To develop a sensitive and reliable assay for detecting enterotoxin genes (nheA, entFM, hblD, cytK) and emetic toxin (ces), specific primers each targeting one individual gene were designed. Propidium monoazide (PMA) was coupled with the developed multiplex PCR (mPCR) for the detection of viable B. cereus. The inclusivity and exclusivity of the PMA-mPCR was confirmed using a panel of 44 strains including 17 emetic and 9 enterotoxic B. cereus reference strains and 18 non-target strains. The limit of detection (LOD) without PMA treatment in pure DNA was 2 pg/reaction tube. The LOD of mPCR assay in pure heat-killed dead bacteria was 4.0 × 102 CFU/mL. Also, the LOD on the viable bacteria with or without PMA treatment was similar (3.8 × 102 CFU/mL) showing that the PMA treatment did not significantly decrease sensitivity. Finally, the newly developed PMA-mPCR successfully detected 4.8 × 103 and 3.6 × 103 CFU/g of viable B. cereus F4810/72 (emetic) and B. cereus ATCC 12480 (enterotoxic) reference strains, respectively, in food samples. Hence, this study combines PMA and mPCR to detect viable B. cereus with a wide range of toxin detection (5 toxins). Thus, the novel PMA-mPCR assay developed in this study is a rapid and efficient diagnostic tool for the monitoring of viable B. cereus in food samples and potentially other samples via appropriate DNA extraction.

Enterotoxigenic profiling of emetic toxin- and enterotoxin-producing Bacillus cereus, Isolated from food, environmental, and clinical samples by multiplex PCR.

Bacillus cereus comprises the largest group of endospore-forming bacteria and can cause emetic and diarrheal food poisoning. A total of 496 B. cereus strains isolated from various sources (food, environmental, clinical) were assessed by a multiplex PCR for the presence of enterotoxin genes. The detection rate of nheA, entFM, hblC, and cytK enterotoxin genes among all B. cereus strains was 92.33%, 77.21%, 59.47%, and 47.58%, respectively. Enterotoxigenic profiles were determined in emetic toxin- (8 patterns) and enterotoxin-producing strains (12 patterns). The results provide important information on toxin prevalence and toxigenic profiles of B. cereus from various sources. Our findings revealed that B. cereus must be considered a serious health hazard and Bacillus thuringiensis should be considered of a greater potential concern to food safety among all B. cereus group members. Also, there is need for intensive and continuous monitoring of products embracing both emetic toxin and enterotoxin genes.

Diversity ofBacillus cereusgroup strains is reflected in their broad range of pathogenicity and diverse ecological lifestyles

Bacillus cereus comprises a highly versatile group of bacteria, which are of particular interest because of their capacity to cause disease. Emetic food poisoning is caused by the toxin cereulide produced during the growth of emetic B.cereus in food, while diarrhoeal food poisoning is the result of enterotoxin production by viable vegetative B.cereus cells in the small intestine, probably in the mucus layer and/or attached to the host's intestinal epithelium. The numbers of B.cereus causing disease are highly variable, depending on diverse factors linked to the host (age, diet, physiology and immunology), bacteria (cellular form, toxin genes and expression) and food (nutritional composition and meal characteristics). Bacillus cereus group strains show impressive ecological diversity, ranging from their saprophytic life cycle in soil to symbiotic (commensal and mutualistic) lifestyles near plant roots and in guts of insects and mammals to various pathogenic ones in diverse insect and mammalian hosts. During all these different ecological lifestyles, their toxins play important roles ranging from providing competitive advantages within microbial communities to inhibition of specific pathogenic organisms for their host and accomplishment of infections by damaging their host's tissues.

Small Bacillus cereus ATCC 14579 subpopulations are responsible for cytotoxin K production

Bacillus cereus diarrhoeal food poisoning can be caused by several potential enterotoxins, including the nonhaemolytic enterotoxin (Nhe), haemolysin BL (Hbl) and cytotoxin K (CytK). To get more insights into the CytK expression, a fluorescent reporter strain was created for CytK expression. Bacillus cereus ATCC 14579 was used as the reporter strain that contained the cyan fluorescent protein (CFPopt) gene under control of the cytK promoter. Transcription of enterotoxin genes nheB, hblC and cytK was assessed by messenger RNA analysis (RT-qPCR), and their full expression was assessed by immunological protein detection in the case of Nhe and Hbl and fluorescence microscopy in the case of CytK, using the reporter gene CFPopt. Transcription of enterotoxins Nhe, Hbl and CytK showed similar kinetics with a peak during the late exponential growth phase. Toxin expression of the reporter strain was unaltered in comparison with the wild type. However, fluorescence, and thus CytK expression, only occurred in a small (1-2%) portion of the cell population. These results suggest that a small subpopulation of B. cereus ATCC 14579 is responsible for CytK production in a homogeneous monoculture. Future research is warranted to determine whether genetically homogeneous B. cereus populations utilize differential gene expression for other toxins and virulence genes than CytK and whether this also applies to other B. cereus strains. If so, differential expression of toxin genes could be used by these bacteria to increase the fitness and survival chances of their population by diversification and specialization into different subpopulations.

Enterotoxin Production byBacillus cereusUnder Gastrointestinal Conditions and Their Immunological Detection by Commercially Available Kits

Currently, three commercial kits for Bacillus cereus enterotoxins Nhe and/or Hbl detection are available, namely, the Bacillus diarrheal enterotoxin visual immunoassay (BDE VIA™) kit (3M Tecra), B. cereus enterotoxin reversed passive latex agglutination (BCET-RPLA) kit (Oxoid), and the Duopath(®) Cereus Enterotoxins (Merck). The performance of the kits and their applicability to gastrointestinal simulation samples were evaluated. Then, the stability and production of enterotoxins Hbl and Nhe under gastrointestinal conditions were investigated. Enterotoxin production was absent or impaired at acidic pH, i.e., in gastric medium with pH 5.0 and lasagne verde with pH 5.5. B. cereus did produce enterotoxins Nhe and Hbl during anaerobic growth in intestinal medium at pH 7.0, but the toxins were instantly degraded by the enzymes in the host's digestive secretions. Preformed enterotoxins did not withstand gastrointestinal passage under the simulated conditions, which suggests that preformed enterotoxins in food do not contribute to the diarrheal food poisoning syndrome. In conclusion, diarrhea is probably caused by de novo enterotoxin production by B. cereus cells located closely to the host's intestinal epithelium.

Inactivation of Bacillus cereus vegetative cells by gastric acid and bile during in vitro gastrointestinal transit

BackgroundThe foodborne pathogen Bacillus cereus can cause diarrhoeal food poisoning by production of enterotoxins in the small intestine. The prerequisite for diarrhoeal disease is thus survival during gastrointestinal passage.MethodsVegetative cells of 3 different B. cereus strains were cultivated in a real composite food matrix, lasagne verde, and their survival during subsequent simulation of gastrointestinal passage was assessed using in vitro experiments simulating transit through the human upper gastrointestinal tract (from mouth to small intestine).ResultsNo survival of vegetative cells was observed, despite the high inoculum levels of 7.0 to 8.0 log CFU/g and the presence of various potentially protective food components. Significant fractions (approx. 10% of the consumed inoculum) of B. cereus vegetative cells survived gastric passage, but they were subsequently inactivated by bile exposure in weakly acidic intestinal medium (pH 5.0). In contrast, the low numbers of spores present (up to 4.0 log spores/g) showed excellent survival and remained viable spores throughout the gastrointestinal passage simulation.ConclusionVegetative cells are inactivated by gastric acid and bile during gastrointestinal passage, while spores are resistant and survive. Therefore, the physiological form (vegetative cells or spores) of the B. cereus consumed determines the subsequent gastrointestinal survival and thus the infective dose, which is expected to be much lower for spores than vegetative cells. No significant differences in gastrointestinal survival ability was found among the different strains. However, considerable strain variability was observed in sporulation tendency during growth in laboratory medium and food, which has important implications for the gastrointestinal survival potential of the different B. cereus strains.

Survival and Germination of Bacillus cereus Spores without Outgrowth or Enterotoxin Production during In Vitro Simulation of Gastrointestinal Transit

To study the gastrointestinal survival and enterotoxin production of the food-borne pathogen Bacillus cereus, an in vitro simulation experiment was developed to mimic gastrointestinal passage in 5 phases: (i) the mouth, (ii) the stomach, with gradual pH decrease and fractional emptying, (iii) the duodenum, with high concentrations of bile and digestive enzymes, (iv) dialysis to ensure bile reabsorption, and (v) the ileum, with competing human intestinal bacteria. Four different B. cereus strains were cultivated and sporulated in mashed potato medium to obtain an inoculum of 7.0 log spores/ml. The spores showed survival and germination during the in vitro simulation of gastrointestinal passage, but vegetative outgrowth of the spores was suppressed by the intestinal bacteria during the final ileum phase. No bacterial proliferation or enterotoxin production was observed, despite the high inoculum levels. Little strain variability was observed: except for the psychrotrophic food isolate, the spores of all strains survived well throughout the gastrointestinal passage. The in vitro simulation experiments investigated the survival and enterotoxin production of B. cereus in the gastrointestinal lumen. The results obtained support the hypothesis that localized interaction of B. cereus with the host's epithelium is required for diarrheal food poisoning.

Application of MALDI-TOF mass spectrometry for the detection of enterotoxins produced by pathogenic strains of the Bacillus cereus group

Enterotoxins produced by different species of the Bacillus cereus group, such as cytotoxin K1 (CytK1) and non-haemolytic enterotoxin (NHE), have been associated with diarrhoeal food poisoning incidents. Detection of CytK1 is not possible with commercial assays while NHE is recognised by an immunological kit (TECRA) that does not specifically target this protein because it is based on polyclonal antibodies. It is evident that the lack of suitable tools for the study of enterotoxins hampers the possibilities for accurate hazard identification and characterisation in microbial food safety risk assessment. We applied matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF/MS) for the detection of CytK1 and NHE produced by pathogenic strains of the B. cereus group using protein digests from 1D gel electrophoresis. Secretion of CytK1 and two of the three components of NHE was confirmed in supernatants of different B. cereus cultures. For each protein, we introduce biomarkers that could be used for the screening of food poisoning or food/environmental isolates that can secrete enterotoxins. For example, tryptic peptides of 2,310.2 and 1,192.5 Da (calculated mass) can be indicators for CytK1 and NheA, respectively, although a simultaneous detection of other enterotoxin-specific peptides is recommended to assure the presence of a toxin in an unknown sample. Comparison of MALDI-TOF/MS with the TECRA kit showed that our methodological strategy performed well and it had the competitive advantage of specifically detecting NheA. Therefore, MALDI-TOF/MS can be successfully incorporated into risk assessment procedures in order to determine the involvement of strains of the B. cereus group in foodborne outbreaks, including the recently described cytK1 producing species, Bacillus cytotoxicus.

Impact of intestinal microbiota and gastrointestinal conditions on the in vitro survival and growth of Bacillus cereus

Ingestion of B. cereus can result in diarrhea, if these bacteria survive gastrointestinal passage and achieve growth and enterotoxin production in the small intestine. The gastrointestinal survival of vegetative cells and spores of the diarrheal food poisoning strain B. cereus NVH 1230-88 was investigated during in vitro batch experiments simulating the stomach, duodenum and ileum using simulation media and competing intestinal microbiota. All spores and approx. 30% of the vegetative B. cereus cells survived the 2h incubation in gastric medium with pH 4.0. Sterile intestinal medium induced germination of spores and enabled outgrowth of vegetative cells to approx. 7 log CFU/mL. The behavior of B. cereus in the intestinal environment with competing intestinal bacteria was determined by their relative concentrations. Besides the numbers of intestinal bacteria, the nutrition and composition of the intestinal community were also very important for the growth inhibition of B. cereus.

Evolution of pathogenicity in the Bacillus cereus group

The Bacillus cereus group of bacteria comprises soil-dwelling saprophytes but on occasion these bacteria can cause a wide range of diseases in humans, including food poisoning, systemic infections and highly lethal forms of anthrax. While anthrax is almost invariably caused by strains from a single evolutionary lineage, Bacillus anthracis, variation in the virulence properties of strains from other lineages has not been fully addressed. Using multi-locus sequence data from 667 strains, we reconstructed the evolutionary history of the B. cereus group in terms of both clonal inheritance and recombination. The strains included 155 clinical isolates representing B. anthracis, and isolates from emetic and diarrhoeal food poisoning, septicaemia and related infections, wound, and lung infections. We confirmed the existence of three major clades and found that clinical isolates of B. cereus (with the exception of emetic toxin-producing strains) are evenly distributed between and within clades 1 and 2. B. anthracis in particular and emetic toxin-producing B. cereus show more clonal structure and are restricted to clade 1. Our characterization of the patterns of genetic exchange showed that there exist partial barriers to gene flow between the three clades. The pathogenic strains do not exhibit atypically high or low rates of recombination, consistent with the opportunistic nature of most pathogenic infections. However, there have been a large number of recent imports in clade 1 of strains from external origins, which is indicative of an on-going shift in gene-flow boundaries for this clade.

Food poisoning potential of Bacillus cereus strains from Norwegian dairies

Characteristics concerning diarrhoeal potential were investigated in B. cereus dairy strains. The thirty-nine strains, isolated from whipping cream, were tested for cytotoxicity after culturing at human body temperature as well as 25 °C and 32 °C. At 37 °C, none of the strains were highly cytotoxic. This observation suggests that those strains should be considered to pose a minor risk with regard to diarrhoeal food poisoning. However, some strains were moderately or highly cytotoxic when grown at 25 °C and 32 °C. While the majority of the strains were able to grow at refrigeration temperatures, only four B. weihenstephanensis strains were identified among them when subjected to discriminative PCR assays and growth temperatures which delimit this species.

Production and characterization of antibodies against each of the three subunits of the Bacillus cereus nonhemolytic enterotoxin complex.

The nonhemolytic enterotoxin (Nhe) is one of the two three-component enterotoxins which are responsible for diarrheal food poisoning syndrome caused by Bacillus cereus. To facilitate the detection of this toxin, consisting of the subunits NheA, NheB, and NheC, a complete set of high-affinity antibodies against each of the three components was established and characterized. A rabbit antiserum specific for the C-terminal part (15 amino acids) of NheC was produced using a respective synthetic peptide coupled to a protein carrier for immunization. Using purified B. cereus exoprotein preparations as immunogens, one monoclonal antibody against NheA and several antibodies against NheB were obtained. No cross-reactivity with other proteins produced by different strains of B. cereus was observed. Antibodies against the NheB component were able to neutralize the cytotoxic activity (up to 98%) of Nhe. Based on indirect enzyme immunoassays, the antibodies developed in this study were successfully used in the characterization of the enterotoxic activity of several B. cereus strains. For the first time, it could be shown that strains carrying the nhe genes usually express the complete set of the three components, including NheC. However, the amount of toxin produced varies considerably between the different strains.

Tripartite haemolysin BL: isolation and characterization of two distinct homologous sets of components from a single Bacillus cereus isolate

Haemolysin BL (HBL), a three-component enterotoxic/necrotizing/vascular permeability toxin, is a likely virulence factor of Bacillus cereus diarrhoeal food poisoning and necrotic infections. This paper describes the isolation of two distinct homologous sets of all three HBL components from a single B. cereus isolate, MGBC 145. The proteins of one set (designated HBL, consisting of B, L(1) and L(2)), were about 87-100% identical in N-terminal amino acid sequences to their respective prototype components from strain F837/76, and the proteins of the homologous set (HBL(a), consisting of B(a), L(1a) and L(2a)) were all about 62-65% identical. Only the latter homologues differed immunochemically and physicochemically from the prototypes. HBL and HBL(a) exhibited similar haemolytic and vascular permeability potencies, and the homologues could be interchanged freely. There were no notable differences in activity between the L component homologues. However, components B and B(a) were significantly different. Both were secreted as monomers, but unlike B, B(a) was isolated as a relatively inactive complex that could be reactivated with urea. When B(a) was substituted for B in gel-diffusion assays the distinct discontinuous haemolysis pattern typical of the presence of B did not occur. In suspension assays, excess B inhibited the haemolysis of B-primed cells by L(1) (as previously described), but not that of B(a)-primed cells. Excess B(a) had the opposite effect and enhanced lysis of B(a)-primed cells, but not that of B-primed cells. These differences reveal details about how the toxin components interact on target cell membranes. The authors' observations indicate that HBL represents a new family of multicomponent toxins that was generated by a process of gene and operon duplication that occurred either intracellularly or by horizontal transfer, and raise the possibility of the existence of other related toxins in the genetically diverse B. cereus taxonomic group.

Insertional inactivation of hblC encoding the L2 component of Bacillus cereus ATCC 14579 haemolysin BL strongly reduces enterotoxigenic activity, but not the haemolytic activity against human erythrocytes.

Haemolysin BL (HBL) is a Bacillus cereus toxin composed of a binding component, B, and two lytic components, L1 and L2. HBL is also the enterotoxin responsible for the diarrhoeal food poisoning syndrome caused by several strains of B. cereus. The three genes encoding the HBL components constitute an operon and are transcribed from a promoter 608 bp upstream of the hblC translational start site. The first gene of the hbl operon, hblC, in the B. cereus type strain, ATCC 14579, was inactivated in this study. Inactivation of hblC strongly reduced both the enterotoxigenic activity of B. cereus ATCC 14579 and the haemolytic activity against sheep erythrocytes, while maintaining full haemolytic activity against human erythrocytes.

Molecular cloning and characterization of the genes encoding the L1 and L2 components of hemolysin BL from Bacillus cereus.

Hemolysin BL, which is composed of a binding component, B, and two lytic components, L1 and L2, is the enterotoxin responsible for the diarrheal food poisoning syndrome caused by strains of Bacillus cereus. To further characterize the toxin, we sought to clone and sequence the genes encoding the L1 and L2 proteins. A genomic library was screened with polyclonal antibody to the L1 and L2 proteins to identify recombinant clones containing the genes. Five clones reacted with the antibody to L2, but none reacted with the antibody to L1. Southern hybridization analysis with oligonucleotide probes designed from the N-terminal amino acid sequences of the L1 and L2 proteins, in conjunction with immunoblot and nucleotide sequence analysis, revealed that the recombinant plasmid from one of the clones contained two genes, hblC and hblD, which encode L2 and L1, respectively. The two genes are arranged in tandem and are separated by only 37 bases. The gene which encodes the B component of hemolysin BL (hblA) is located immediately downstream from the gene encoding the L1 protein. Northern blot analysis of B. cereus RNA showed a 5.5-kb transcript which hybridized with DNA fragments internal to, or including a portion of, the coding sequences of the B, L1, and L2 genes, suggesting that the clustered genes which encode the components of hemolysin BL are cotranscribed and constitute an operon.

Enterotoxic activity of hemolysin BL from Bacillus cereus.

Bacillus cereus causes exotoxin-mediated diarrheal food poisoning. Hemolysin BL (HBL) is a well-characterized B. cereus toxin composed of three components (B, L1, and L2) that together possess hemolytic, cytotoxic, dermonecrotic, and vascular permeability activities. Here, we show that HBL causes fluid accumulation in ligated rabbit ileal loops at a dose of 5 micrograms of each component per loop. Maximal fluid responses occurred for combinations of all three components at > or = 25 micrograms of each component per loop. Individual components and binary combinations did not cause significant fluid accumulation at 25 micrograms of each component. Specific antisera to HBL components inhibited the fluid accumulation response of crude culture supernatant from B. cereus F837/76. These antisera were tested against an antiserum to a partially characterized multicomponent diarrheal toxin described previously by Thompson et al. (N. E. Thompson, M. J. Ketterhagen, M. S. Bergdoll, and E. J. Shantz, Infect. Immun. 43:887-894, 1984). Immunoblot and immunoprecipitation analyses indicate that HBL and that toxin are identical. These results confirm previous speculation that HBL is a tripartite enterotoxin that, as for all of its other known activities, requires all three components for maximal activity.