Porcine Gastric Mucin Triggers Toxin Production of Enteropathogenic Bacillus cereus.

Enteropathogenic Bacillus cereus causes foodborne infections due to the production of pore-forming enterotoxins in the intestine. Before that, spores have to be ingested, survive the stomach passage, and germinate. Thus, before reaching epithelial cells, B. cereus comes in contact with the intestinal mucus layer. In the present study, different aspects of this interaction were analyzed. Total RNA sequencing revealed major transcriptional changes of B. cereus strain F837/76 upon incubation with porcine gastric mucin (PGM), comprising genes encoding enterotoxins and further putative virulence factors, as well as proteins involved in adhesion to and degradation of mucin. Indeed, PGM was partially degraded by B. cereus via secreted, EDTA-sensitive proteases. The amount of enterotoxins detectable in culture media supplemented with PGM was also clearly increased. Tests of further strains revealed that enhancement of enterotoxin production upon contact with PGM is broadly distributed among B. cereus strains. Interestingly, evidence was found that PGM can also strain-specifically trigger germination of B. cereus spores and that vegetative cells actively move toward mucin. Overall, our data suggest that B. cereus is well adapted to the host environment due to massive transcriptome changes upon contact with PGM, attributing mucin an important and, thus far, neglected role in pathogenesis.

Multifaceted toxin profile, an approach toward a better understanding of probiotic Bacillus cereus.

Strains of the Bacillus cereus group have been widely used as probiotics for human beings, food animals, plants, and environmental remediation. Paradoxically, B. cereus is responsible for both gastrointestinal and nongastrointestinal syndromes and represents an important opportunistic food-borne pathogen. Toxicity assessment is a fundamental issue to evaluate safety of probiotics. Here, we summarize the state of our current knowledge about the toxins of B. cereus sensu lato to be considered for safety assessment of probiotic candidates. Surfactin-like emetic toxin (cereulide) and various enterotoxins including nonhemolytic enterotoxin, hemolysin BL, and cytotoxin K are responsible for food poisoning outbreaks characterized by emesis and diarrhea. In addition, other factors, such as hemolysin II, Certhrax, immune inhibitor A1, and sphingomyelinase, contribute to toxicity and overall virulence of B. cereus.

Identification of cereulide producing Bacillus cereus by MALDI-TOF MS.

The Bacillus (B.) cereus group consists of nine recognized species which are present worldwide. B. cereus play an important role in food-borne diseases by producing different toxins. Yet, only a small percentage of B. cereus strains are able to produce the heat stable cereulide, the causative agent of emetic food poisoning. To minimize the entry of emetic B. cereus into the food chain, food business operators are dependent on efficient and reliable methods enabling the differentiation between emetic and non-emetic strains. Currently, only time-consuming cell bioassays, molecular methods and tandem mass spectrometry are available for this purpose. Thus, the aim of the present study was to establish a fast and reliable method for the differentiation between emetic/non-emetic strains by MALDI-TOF MS. Selected strains/isolates of the B. cereus group as well as other Bacillus spp. (total n = 121) were cultured on sheep blood agar for 48 h before analysis. Subsequently, the cultures were directly analyzed by MALDI-TOF MS without prior extraction steps. The samples were measured in the mass range of m/z 800-1800 Da. Using ClinProTools 3.0 statistical software and Flex analysis software (Bruker Daltonics GmbH, Bremen, Germany), a differentiation between emetic/non-emetic isolates was possible with a rate of correct identification of 99.1% by means of the evaluation of two specific biomarkers (m/z 1171 and 1187 Da).

Assessing the toxic potential of enteropathogenic Bacillus cereus.

The diarrheal type of food poisoning caused by enteropathogenic Bacillus cereus has been linked to various exotoxins. Best described are the non-hemolytic enterotoxin (Nhe), hemolysin BL (Hbl), and cytotoxin K (CytK). Due to the ubiquitous prevalence of B. cereus in soil and crops and its ability to form highly resistant endospores, contaminations during food production and processing cannot be completely avoided. Although phylogenetically closely related, enteropathogenic B. cereus strains show a high versatility of their toxic potential. Thus, functional tools for evaluating the pathogenic potential are urgently needed in order to predict hazardous food contaminations. As the diarrheal syndrome is the result of a toxico-infection with enterotoxin production in the intestine, the entire passage of the bacteria within the host, from spore survival in the stomach, spore germination, host cell adherence, and motility, to enterotoxin production under simulated intestinal conditions was compared in a panel of 20 strains, including high pathogenic as well as apathogenic ones. This approach resulted in an overarching virulence analysis scheme. In parallel, we searched for potential toxico-specific secreted markers to discriminate low and high pathogenic strains. To this end, we targeted known exotoxins using an easy to implement immunoblotting approach as well as a caseinolytic exoprotease activity assay. Overall, Nhe component B, sphingomyelinase, and exoproteases showed good correlation with the complex virulence analysis scheme and can serve as a template for future fast and easy risk assessment tools to be implemented in routine diagnostic procedures and HACCP studies.

Non-hemolytic enterotoxin of Bacillus cereus induces apoptosis in Vero cells.

Bacillus cereus is an opportunistic pathogen that often causes foodborne infectious diseases and food poisoning. Non-hemolytic enterotoxin (Nhe) is the major toxin found in almost all enteropathogenic B. cereus and B. thuringiensis isolates. However, little is known about the cellular response after Nhe triggered pore formation on cell membrane. Here, we demonstrate that Nhe induced cell cycle arrest at G0 /G1 phase and provoked apoptosis in Vero cells, most likely associated with mitogen-activated protein kinase (MAPK) and death receptor pathways. The influx of extracellular calcium ions and increased level of reactive oxygen species in cytoplasm were sensed by apoptosis signal-regulating kinase 1 (ASK1) and p38 MAPK. Extrinsic death receptor Fas could also promote the activation of p38 MAPK. Subsequently, ASK1 and p38 MAPK triggered downstream caspase-8 and 3 to initiate apoptosis. Our results clearly demonstrate that ASK1, and Fas-p38 MAPK-mediated caspase-8 dependent pathways are involved in apoptotic cell death provoked by the pore-forming enterotoxin Nhe.

A Biosurfactant-Inspired Heptapeptide with Improved Specificity to Kill MRSA.

The emergence and rapid spread of methicillin-resistant Staphylococcus aureus (MRSA) poses a serious threat to public health. New antibiotics and strategies are urgently needed to combat S. aureus associated infections. Bacaucin, a novel cyclic lipopeptide from Bacillus subtilis CAU21, is reported. Bacaucin shows broad antibacterial activity against Gram-positive bacteria, but is also hemolytic and cytotoxic. However, bacaucin-1, a bacaucin-inspired ring-opened heptapeptide, shows specific antibacterial activity against MRSA by a membrane-disruptive mechanism without detectable toxicity to mammalian cells or induction of bacterial resistance. Bacaucin-1 was efficient in preventing infections in both in vitro and in vivo models and is a valuable prototype antibiotic with high potential against S. aureus infections.

Formation of small transmembrane pores: An intermediate stage on the way to Bacillus cereus non-hemolytic enterotoxin (Nhe) full pores in the absence of NheA.

The non-hemolytic enterotoxin (Nhe) of Bacillus cereus is a three-partite toxin formed of the components NheA, -B and -C. Pore formation and subsequent lysis of target cells caused by Nhe is an orchestrated process comprising three steps: (i) formation of NheB/C oligomers in solution, (ii) attachment of the oligomers to the cell membrane, (iii) binding of NheA to the oligomers. The present study aimed to characterize the properties of the NheB/C complex and the fate of the target cell upon binding. An enzyme immunoassay allowing kinetic measurements and surface plasmon resonance revealed the fast and high affinity formation of the NheB/C oligomers. The benefit of these complexes is a more stable cell binding as well as stronger and earlier cytotoxic effect. High molecular mass hetero-oligomers (620 kDa) probably consisting of one NheC and up to 15 NheB were detected by size-exclusion chromatography and on native PAGE immunoblots. Due to the NheBC application the morphology and membrane permeability of Vero cells is partly disturbed. Formation of stable transmembrane channels with a conductance of about 870 pS and a diameter of about 2 nm due to the application of NheBC could be demonstrated in lipid bilayer experiments. Thus, the NheBC complex itself has a tendency to increase the membrane permeability prior to the emergence of full pores containing also NheA.

Simultaneous Rapid Detection and Serotyping of Cronobacter sakazakii Serotypes O1, O2, and O3 by Using Specific Monoclonal Antibodies.

Cronobacter sakazakii is a foodborne pathogen associated with rare but often lethal infections in neonates. Powdered infant formula (PIF) represents the most frequent source of infection. Out of the identified serotypes (O1 to O7), O1, O2, and O3 are often isolated from clinical and PIF samples. Serotype-specific monoclonal antibodies (MAbs) suitable for application in enzyme immunoassays (EIAs) for the rapid detection of C. sakazakii have not yet been developed. In this study, we created specific MAbs with the ability to bind toC. sakazakii of serotypes O1, O2, and O3. Characterization by indirect EIAs, immunofluorescence, motility assays, and immunoblotting identified lipopolysaccharide (LPS) and exopolysaccharide (EPS) as the antigenic determinants of the MAbs. The established sandwich EIAs were highly sensitive and were able to detect between 2 × 10(3)and 9 × 10(6)CFU/ml. Inclusivity tests confirmed that 93% of serotype O1 strains, 100% of O2 strains, and 87% of O3 strains were detected at low cell counts. No cross-reactivity with >100 strains of Cronobacter spp. and other Enterobacter iaceae was observed, except for that with C. sakazakii serotype O3 and Cronobacter muytjensii serotype O1. Moreover, the sandwich EIAs detected C. sakazakii in PIF samples artificially contaminated with 1 to 10 bacterial cells per 10 g of sample after 15 h of preenrichment. The use of these serotype-specific MAbs not only allows the reliable detection of C. sakazakii strains but also enables simultaneous serotyping in a simple sandwich EIA method.

A Cronobacter turicensis O1 antigen-specific monoclonal antibody inhibits bacterial motility and entry into epithelial cells.

Cronobacter turicensis is an opportunistic foodborne pathogen that can cause a rare but sometimes lethal infection in neonates. Little is known about the virulence mechanisms and intracellular lifestyle of this pathogen. In this study, we developed an IgG monoclonal antibody (MAb; MAb 2G4) that specifically recognizes the O1 antigen of C. turicensis cells. The antilipopolysaccharide antibody bound predominantly monovalently to the O antigen and reduced bacterial growth without causing cell agglutination. Furthermore, binding of the antibody to the O1 antigen of C. turicensis cells caused a significant reduction of the membrane potential which is required to energize flagellar rotation, accompanied by a decreased flagellum-based motility. These results indicate that binding of IgG to the O antigen of C. turicensis causes a direct antimicrobial effect. In addition, this feature of the antibody enabled new insight into the pathogenicity of C. turicensis. In a tissue culture infection model, pretreatment of C. turicensis with MAb 2G4 showed no difference in adhesion to human epithelial cells, whereas invasion of bacteria into Caco-2 cells was significantly inhibited.

First Insights into the Occurrence of Circular Single-Stranded DNA Genomes in Asian and African Cattle.

Circular replicase-encoding single-stranded (CRESS) DNA viruses and other circular DNA agents are increasingly found in various samples and animals. A specific class of these agents-termed bovine meat and milk factors (BMMF)-has been supposed to act as a factor in indirect carcinogenesis in humans. Initial observations attributed the BMMF to European cattle breeds and foodstuffs produced thereof. In the present study, blood and fecal samples from African and Asian cattle were examined. BMMF molecules and genomoviruses were detected in all bovids under study. The majority (79%) of the 29 circular elements could be assigned to BMMF groups 1 and 2, whereas CRESS viruses of the family Genomoviridae accounted for the smaller part (21%). Two genomoviruses belong to the genus Gemykibivirus and one to the genus Gemykrogvirus. The remaining three might be considered as novel species within the genus Gemycircularvirus. The majority of all isolated molecules originated from fecal samples, whereas only three derived from blood. The results from this study expand our knowledge on the diversity and presence of circular DNA in different ruminants that serve for food production in many countries over the world.

Monoclonal antibodies neutralize Bacillus cereus Nhe enterotoxin by inhibiting ordered binding of its three exoprotein components.

The Nhe enterotoxin from Bacillus cereus is known to induce cytotoxicity on Vero and CaCo-2 cells by ordered binding of its single components NheA, NheB, and NheC. This study aimed to elucidate functional sites on NheB by identifying the epitopes of the neutralizing monoclonal antibodies 1E11 and 2B11. The binding regions of both antibodies were determined by using recombinant NheB fragments and synthetic peptides. The antigenic site of antibody 1E11 was located within the amino acids 321 to 341 of NheB, whereas reactivity of antibody 2B11 was dependent on the presence of amino acids 122 to 150 and on conformation. Both antibodies were able to bind simultaneously to NheB and did not interfere with target cell binding as shown by immunofluorescence microscopy. A set of neutralization assays revealed that antibody 2B11 most likely interfered with the interaction between NheB and NheC both on the epithelium cell surface and in solution. In contrast, antibody 1E11 inhibited association between NheA and cell-bound NheB in a competitive manner, and effectively neutralized Nhe cytotoxicity on a variety of human cell lines. This distinct mechanism further supports that NheA is the key component during the Nhe mode of action and the C-terminal epitope recognized by antibody 1E11 points to an important functional region of NheB.

A broadly applicable approach to prepare monoclonal anti-cephalosporin antibodies for immunochemical residue determination in milk.

A simple, efficient and rapid method for the synthesis of cephalosporin-protein conjugates was established. These conjugates were used as immunogens to produce monoclonal antibodies (mAbs) and as solid phase antigens in competitive indirect enzyme immunoassays (EIAs). With this generic approach, a novel set of monoclonal antibodies for cephalosporins was prepared, including ceftiofur and cephalexin as well as, reported here for the first time, cefoperazone, cefquinome and cephapirin. All 5 EIAs were highly sensitive, with standard curve IC(50) values of 0.7 (ceftiofur), 1.1 (cefquinome), 5.2 (cephalexin), 13.8 (cefoperazone) and 40.3 ng mL(-1) (cephapirin). Detection limits (IC(30)) ranged from 0.3 (ceftiofur mAb 1D7) to 17.2 ng mL(-1) (cephapirin mAb 2F10). Specificity studies revealed that cephalosporin-antibody binding was strongly determined by the side chain residues of the cephem nucleus. Therefore all mAbs, to some extent, recognized other beta-lactam antibiotics with similar side chain residues. Within the group of cephalosporins approved for use in veterinary medicine, however, the final EIAs were highly selective for their respective antigen, except for the ceftiofur EIA which showed cross-reactions with cefquinome. The applicability of the five assays for drug residue testing in milk was demonstrated. In each EIA the target drug could be determined in milk with high accuracy and precision at concentrations far below the European Union maximum residue limits.

Characterization of strain-specific Bacillus cereus swimming motility and flagella by means of specific antibodies.

One of the multiple factors determining the onset of the diarrhoeal disease caused by enteropathogenic Bacillus cereus is the ability of the bacteria to actively move towards the site of infection. This ability depends on flagella, but it also varies widely between different strains. To gain more insights into these strain-specific variations, polyclonal rabbit antisera as well as monoclonal antibodies (mAbs) were generated in this study, which detected recombinant and natural B. cereus flagellin proteins in Western blots as well as in enzyme immunoassays (EIAs). Based on mAb 1A11 and HRP-labelled rabbit serum, a highly specific sandwich EIA was developed. Overall, it could be shown that strain-specific swimming motility correlates with the presence of flagella/flagellin titres obtained in EIAs. Interestingly, mAb 1A11, recognizing an epitope in the N-terminal region of the flagellin protein, proved to inhibit bacterial swimming motility, while the rabbit serum rather decreased growth of selected B. cereus strains. Altogether, powerful tools enabling the in-depth characterization of the strain-specific variations in B. cereus swimming motility were developed.

Penetration of Enrofloxacin in Aqueous Humour of Avian Eyes.

Enrofloxacin has been shown to be appropriate to treat bacterial eye infections in mammals. However, the anatomy and physiology of the avian eye substantially differ from those in mammals, and pharmacokinetic data substantiating the clinical efficacy of enrofloxacin in birds are still lacking. In total, 40 chickens (Gallus gallus, Lohman Selected Leghorn) received single intramuscular administration of enrofloxacin at a dosage of 25 mg/kg body weight (BW). Serial blood and aqueous humour samples were taken at 12 different time points after administration (0-60 min and 2-32 h) and were analysed for their fluoroquinolone concentrations using a competitive enzyme immunoassay. The metabolization of enrofloxacin to ciprofloxacin was determined using liquid chromatography-mass spectrometry. The maximum serum concentrations of fluoroquinolones were observed at the time point of 2.82 ± 0.1 h and amounted to 10.67 ± 0.5 µg/mL. Fluoroquinolones redistributed to a minor extent into the aqueous humour reaching maximum concentrations of 4.52 ± 1.2 µg/mL after 7.54 ± 1.0 h of drug administration. The mean residence time (MRT), volume of distribution (Vd), and terminal half-life (t1/2 ß) were 1.68-, 2.84-, and 2.01-fold higher in aqueous humour than in serum, indicating that fluoroquinolones were trapped in aqueous humour. Enrofloxacin was only marginally metabolized into ciprofloxacin. A single intramuscular injection of a therapeutical dose of enrofloxacin (25 mg/kg BW) thus generated sustained and therapeutically active levels of enrofloxacin in the aqueous humour of chicken eyes.

Pudding Proteomics: Cyclomaltodextrin Glucanotransferase and Microbial Proteases Can Liquefy Extended Shelf Life Dairy Products.

In recent years, a lack of stability of dairy products with extended shelf life (e.g., yoghurt products, UHT desserts) has occurred, with the corresponding products liquefying significantly after days or weeks. This project aimed to identify the enzymes responsible for the liquefaction of the affected products based on differential proteomic analyses. No evidence was found for the presence of starch-degrading bacteria in the affected products. With zymography and proteome analysis, we detected the cause of liquefaction in a pudding by contamination of its aroma component with an engineered amylolytic enzyme, cyclomaltodextrin glucanotransferase (CGTase) from Thermoanaerobacterium thermosulfurigenes. In addition, we detected contamination with Pseudomonas-derived proteolytic ATP-dependent Clp protease in one pudding batch and proteases in technically used amylases, which degraded ß-caseins in another batch. Identification of these agents with liquefying properties in dairy products are useful for adjustment of production protocols and/or composition of additives, and thus shelf life extension.

The Food Poisoning Toxins of Bacillus cereus.

Bacillus cereus is a ubiquitous soil bacterium responsible for two types of food-associated gastrointestinal diseases. While the emetic type, a food intoxication, manifests in nausea and vomiting, food infections with enteropathogenic strains cause diarrhea and abdominal pain. Causative toxins are the cyclic dodecadepsipeptide cereulide, and the proteinaceous enterotoxins hemolysin BL (Hbl), nonhemolytic enterotoxin (Nhe) and cytotoxin K (CytK), respectively. This review covers the current knowledge on distribution and genetic organization of the toxin genes, as well as mechanisms of enterotoxin gene regulation and toxin secretion. In this context, the exceptionally high variability of toxin production between single strains is highlighted. In addition, the mode of action of the pore-forming enterotoxins and their effect on target cells is described in detail. The main focus of this review are the two tripartite enterotoxin complexes Hbl and Nhe, but the latest findings on cereulide and CytK are also presented, as well as methods for toxin detection, and the contribution of further putative virulence factors to the diarrheal disease.

Cytotoxicity of the Bacillus cereus Nhe enterotoxin requires specific binding order of its three exoprotein components.

This study focuses on the interaction of the three components of the Bacillus cereus Nhe enterotoxin with particular emphasis on the functional roles of NheB and NheC. The results demonstrated that both NheB and NheC were able to bind to Vero cells directly while NheA lacked this ability. It was also shown that Nhe-induced cytotoxicity required a specific binding order of the individual components whereby the presence of NheC in the priming step as well as the presence of NheA in the final incubation step was mandatory. Priming of cells with NheB alone and addition of NheA plus NheC in the second step failed to induce toxic effects. Furthermore, in solution, excess NheC inhibited binding of NheB to Vero cells, whereas priming of cells with excess NheC resulted in full toxicity if unbound NheC was removed before addition of NheB. By using mutated NheC proteins where the two cysteine residues in the predicted beta-tongue were replaced with glycine (NheCcys-) or where the entire hydrophobic stretch was deleted (NheChr-), the predicted hydrophobic beta-tongue of NheC was found essential for binding to cell membranes but not for interaction with NheB in solution. All data presented here are compatible with the following model. The first step in the mode of action of Nhe is associated with binding of NheC and NheB to the cell surface and probably accompanied by conformational changes. These events allow subsequent binding of NheA, leading to cell lysis.

Detection of Bacillus cereus with enteropathogenic potential by multiplex real-time PCR based on SYBR Green I.

In order to meet the growing demand for fast and reliable detection of potentially toxinogenic Bacillus cereus, we developed a multiplex real-time PCR assay based on SYBR Green I with subsequent melting curve analysis. We designed and selected primers specific for genes of toxins responsible for diarrhoea (nheA, hblD and cytK1) and emesis (ces). A panel of 337 Bacillus strains was applied to the novel method on Light Cycler 2.0 with average melting temperature (T(m)) values of 73.85 degrees C (nheA), 87.01 degrees C (hblD), 78.66 degrees C (ces) and 82.19 degrees C (cytK1). An adapted version of the assay was also successfully run on Light Cycler 480 using one third (113 strains) of the total test panel. Verification of PCR results by conventional PCR as well as immunoassays and cytotoxicity tests gave an overall excellent correlation. Distinct melting peaks were only observed in B. cereus and B. cereus group strains but not in other Bacilli and Gram-positive or Gram-negative bacteria. Artificial contamination of three different food matrices with distinct bacterial counts revealed a detection limit of 10(1) CFU/g B. cereus cells after overnight enrichment. Thus, the novel multiplex real-time PCR turned out to be a reliable method for identification of B. cereus with enteropathogenic potential.

An immunochemical test for rapid screening of zearalenone and T-2 toxin.

An immunochemically based test for non-instrumental simultaneous detection of zearalenone (ZEA) and T-2 toxin (T2) in feed was developed. The method combines clean-up of sample extract, pre-concentration of analytes by immunoextraction and immunodetection through the enzymatic reaction of horseradish peroxidase (HRP). The test is housed inside a standard 1-mL solid-phase extraction column and consists of three layers: two test layers (one for ZEA and another for T2) with immobilised specific antibodies and one control layer with bound anti-HRP antibodies. Feed extract was passed through an additional column with clean-up layer, which was disconnected after extract application. Total assay time was about 15 min for six samples and detection time was 4 min after chromogenic substrate application. Under optimised conditions a cut-off level for ZEA and T2 of 100 microg/kg was established. Different feed types were analysed for ZEA and T2 contamination by the proposed method and results were confirmed by LC-MS/MS.