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Monika Ehlingschulz - One of the best experts on this subject based on the ideXlab platform.

  • why be serious about emetic bacillus cereus Cereulide production and industrial challenges
    Food Microbiology, 2020
    Co-Authors: Katia Rouzeauszynalski, Katharina Stollewerk, Ute Messelhausser, Monika Ehlingschulz
    Abstract:

    Abstract Cereulide, a potent toxin produced by Bacillus cereus, is a small, highly heat- and acid- resistant depsipeptide toxin, which confronts food industry with several challenges. Due to the ubiquitous presence of B. cereus in the environment, this opportunistic pathogen can enter food production and processing at almost any stage. Although the bacteria itself might be removed during food processing, the Cereulide toxin will most likely not be destroyed or inactivated by these processes. Because of the high toxicity of Cereulide and the high incidence rates often observed in connection with foodborne outbreaks, the understanding of the mechanisms of toxin production as well as accurate data on contamination sources and factors promoting toxin formation are urgently needed to prevent contamination and toxin production in food production processes. Over the last decade, considerable progress had been made on the understanding of Cereulide toxin biosynthesis in emetic B. cereus, but an overview of current knowledge on this toxin with regards to food industry perspective is lacking. Thus, we aim in this work to summarize data available on extrinsic parameters acting on Cereulide toxin synthesis in emetic B. cereus and to discuss the food industry specific challenges related to this toxin. Furthermore, we emphasize how identification of the cardinals in food production processes can lead to novel effective strategies for prevention of toxin formation in the food processing chain and could contribute to the improvement of existing HACCP studies.

  • evaluation of maldi tof mass spectrometry for rapid detection of Cereulide from bacillus cereus cultures
    bioRxiv, 2019
    Co-Authors: Joerg Doellinger, Monika Ehlingschulz, Timo D Stark, Andy Schneider, Peter Lasch
    Abstract:

    Bacillus cereus plays an often unrecognized role in food borne diseases. Food poisoning caused by this pathogen is manifested by either diarrhea or emesis. While different enterotoxins have been linked to the diarrheal type of B. cereus infections, the emetic toxin Cereulide is responsible for the second type. Due to the relatively high prevalence of Cereulide associated food poisoning, methods for simple and reliable detection of Cereulide producing strains are of utmost importance. Currently, liquid-chromatography coupled to mass spectrometry (LC-MS) is used for sensitive, specific and quantitative Cereulide detection, but this technique requires specialized LC-MS equipment, which is often not available in microbiology routine diagnostic laboratories. The last decade has witnessed the advent of matrix-assisted laser desorption/ionization-time-of flight mass spectrometry (MALDI-ToF MS) as a simple, rapid and cost-efficient technique for identification of microbial pathogens in routine diagnostics. Just recently, two different studies reported on the application of MALDI-ToF MS for either the differentiation of emetic and non-emetic strains of B. cereus or for direct detection of Cereulide from bacterial colony smears. However, no method evaluation and optimization was performed in frame of these studies. Thus, additional investigations on the selectivity and sensitivity of MALDI-TOF MS for Cereulide detection are needed before implementation of this method in routine diagnostics can be considered. These aspects prompted us to investigate open or controversial issues and to systematically test sample preparation methods, commonly used for microbial identification for their suitability to detect the emetic toxin directly from bacteria. Based on our experimental findings we propose a MALDI-ToF MS workflow that allows identification of B. cereus and sensitive detection of Cereulide in parallel, using standard, linear-mode MALDI-ToF MS equipment. The experimental protocol is based on the well-established ethanol/formic acid extraction method and offers, if required, possibilities for further characterization by more sophisticated LC-MS-based methods. In summary, the ease of use and the achieved level of analytical sensitivity as well as the wide-spread availability of standard MALDI-ToF MS equipment in clinical microbiological laboratories provides a promising tool to improve and to facilitate routine diagnostics of B. cereus associated food intoxications.

  • first insights into within host translocation of the bacillus cereus toxin Cereulide using a porcine model
    Frontiers in Microbiology, 2018
    Co-Authors: Tobias Bauer, Wolfgang Sipos, Timo D Stark, Tobias Kaser, Christian Knecht, Rene Brunthaler, Armin Saalmuller, Thomas Hofmann, Monika Ehlingschulz
    Abstract:

    Bacillus cereus is a gram-positive pathogen mainly known to evoke two types of foodborne poisonings. The diarrheal syndrome is caused by enterotoxins produced during growth in the intestine. In contrast, the emetic type is caused by the dodecadepsipeptide Cereulide pre-formed in food. Usually, both diseases are self-limiting but occasionally more severe forms, including fatal ones, are reported. Since the mechanisms of Cereulide toxin uptake and translocation within the body as well as the mechanism of its toxic action are still unknown, we used a porcine model to investigate the uptake, routes of excretion and distribution of Cereulide within the host. Pigs were orally challenged with Cereulide using single doses of 10 to 150 µg Cereulide kg-1 body weight to study acute effects or using daily doses of 10 µg Cereulide kg-1 body weight administered for 7 days to investigate effects of longtime, chronic expose. Our study showed that part of Cereulide ingested with food is rapidly excreted with feces while part of the Cereulide toxin is absorbed, passes through membranes and is distributed within the body. Results from the chronic trial indicate bioaccumulation of Cereulide in certain tissues and organs, such as kidney, liver, muscles and fat tissues. Beside its detection in various tissues and organs, our study also demonstrated that Cereulide is able to cross the blood-brain-barrier, which may partially explain the cerebral effects reported from human intoxication cases. The neurobehavioral symptoms, such as seizures and lethargy, observed in our porcine model resemble those reported from human food borne intoxications. The rapid onset of these symptoms indicates direct effects of Cereulide on the central nervous system (CNS), which warrant further research. The porcine model presented here might be useful to study the specific neurobiological effect in detail. Furthermore, our study revealed that typical diagnostic specimens used in human medicine, such as blood samples and urine, are not suitable for diagnostics of food borne Cereulide intoxications. Instead, screening of fecal samples by SIDA-LC-MS may represent a simple and non-invasive method for detection of Cereulide intoxications in clinical settings as well as in foodborne outbreak situations.

  • ces locus embedded proteins control the non ribosomal synthesis of the Cereulide toxin in emetic bacillus cereus on multiple levels
    Frontiers in Microbiology, 2015
    Co-Authors: Genia Lucking, Elrike Frenzel, Siegfried Scherer, Timo D Stark, Thomas Hofmann, Sandra Marxen, Andrea Rütschle, Monika Ehlingschulz
    Abstract:

    The emetic toxin Cereulide produced by Bacillus cereus is synthesized by the modular enzyme complex Ces that is encoded on a pXO1-like megaplasmid. To decipher the role of the genes adjacent to the structural genes cesA/cesB, coding for the non-ribosomal peptide synthetase (NRPS), gene inactivation- and overexpression mutants of the emetic strain F4810/72 were constructed and their impact on Cereulide biosynthesis was assessed. The hydrolase CesH turned out to be a part of the complex regulatory network controlling Cereulide synthesis on a transcriptional level, while the ABC transporter CesCD was found to be essential for post-translational control of Cereulide synthesis. Using a gene inactivation approach, we show that the NRPS activating function of the phosphopantetheinyl transferase (PPtase) embedded in the ces locus was complemented by a chromosomally encoded Sfp-like PPtase, representing an interesting example for the functional interaction between a plasmid encoded NRPS and a chromosomally encoded activation enzyme. In summary, our results highlight the complexity of Cereulide biosynthesis and reveal multiple levels of toxin formation control. ces operon internal genes were shown to play a pivotal role by acting at different levels of toxin production, thus complementing the action of the chromosomal key transcriptional regulators AbrB and CodY.

  • multiparametric quantitation of the bacillus cereus toxins Cereulide and isoCereulides a g in foods
    Journal of Agricultural and Food Chemistry, 2015
    Co-Authors: Sandra Marxen, Elrike Frenzel, Monika Ehlingschulz, Genia Lucking, Siegfried Scherer, Timo D Stark, Andrea Rütschle, Thomas Hofmann
    Abstract:

    Consumption of food products contaminated with Cereulide (1), a toxin produced by Bacillus cereus, might cause intoxications with symptoms reported to range from indigestion pain and emesis to death. Recently, a series of structural variants, coined isoCereulides A–G (2–8), were identified for the first time to be produced along with Cereulide (1). The observation that isoCereulide A (2) shows an ∼8-fold increased cytotoxicity when compared to 1 urges the development of analytical tools enabling an accurate quantitation of these toxins. Therefore, a rapid, sensitive, and robust stable isotope dilution assay (SIDA) was developed for the combined quantitation of 1–8 by means of UPLC-MS/MS. On average, trueness and precision of the method were 112.5 ± 1.8% RSD, repeatability and reproducibility were 2 and 4% for Cereulide and isoCereulides A–G, and the LOD and LOQ of 0.1 and 0.5 ng/g, respectively, demonstrated a high sensitivity for the developed SIDA method. Application of this method to food samples revea...

Siegfried Scherer - One of the best experts on this subject based on the ideXlab platform.

  • ces locus embedded proteins control the non ribosomal synthesis of the Cereulide toxin in emetic bacillus cereus on multiple levels
    Frontiers in Microbiology, 2015
    Co-Authors: Genia Lucking, Elrike Frenzel, Siegfried Scherer, Timo D Stark, Thomas Hofmann, Sandra Marxen, Andrea Rütschle, Monika Ehlingschulz
    Abstract:

    The emetic toxin Cereulide produced by Bacillus cereus is synthesized by the modular enzyme complex Ces that is encoded on a pXO1-like megaplasmid. To decipher the role of the genes adjacent to the structural genes cesA/cesB, coding for the non-ribosomal peptide synthetase (NRPS), gene inactivation- and overexpression mutants of the emetic strain F4810/72 were constructed and their impact on Cereulide biosynthesis was assessed. The hydrolase CesH turned out to be a part of the complex regulatory network controlling Cereulide synthesis on a transcriptional level, while the ABC transporter CesCD was found to be essential for post-translational control of Cereulide synthesis. Using a gene inactivation approach, we show that the NRPS activating function of the phosphopantetheinyl transferase (PPtase) embedded in the ces locus was complemented by a chromosomally encoded Sfp-like PPtase, representing an interesting example for the functional interaction between a plasmid encoded NRPS and a chromosomally encoded activation enzyme. In summary, our results highlight the complexity of Cereulide biosynthesis and reveal multiple levels of toxin formation control. ces operon internal genes were shown to play a pivotal role by acting at different levels of toxin production, thus complementing the action of the chromosomal key transcriptional regulators AbrB and CodY.

  • multiparametric quantitation of the bacillus cereus toxins Cereulide and isoCereulides a g in foods
    Journal of Agricultural and Food Chemistry, 2015
    Co-Authors: Sandra Marxen, Elrike Frenzel, Monika Ehlingschulz, Genia Lucking, Siegfried Scherer, Timo D Stark, Andrea Rütschle, Thomas Hofmann
    Abstract:

    Consumption of food products contaminated with Cereulide (1), a toxin produced by Bacillus cereus, might cause intoxications with symptoms reported to range from indigestion pain and emesis to death. Recently, a series of structural variants, coined isoCereulides A–G (2–8), were identified for the first time to be produced along with Cereulide (1). The observation that isoCereulide A (2) shows an ∼8-fold increased cytotoxicity when compared to 1 urges the development of analytical tools enabling an accurate quantitation of these toxins. Therefore, a rapid, sensitive, and robust stable isotope dilution assay (SIDA) was developed for the combined quantitation of 1–8 by means of UPLC-MS/MS. On average, trueness and precision of the method were 112.5 ± 1.8% RSD, repeatability and reproducibility were 2 and 4% for Cereulide and isoCereulides A–G, and the LOD and LOQ of 0.1 and 0.5 ng/g, respectively, demonstrated a high sensitivity for the developed SIDA method. Application of this method to food samples revea...

  • depsipeptide intermediates interrogate proposed biosynthesis of Cereulide the emetic toxin of bacillus cereus
    Scientific Reports, 2015
    Co-Authors: Sandra Marxen, Elrike Frenzel, Monika Ehlingschulz, Genia Lucking, Siegfried Scherer, Timo D Stark, Andrea Rütschle, Thomas Hofmann
    Abstract:

    Cereulide and isoCereulides A-G are biosynthesized as emetic toxins by Bacillus cereus via a non-ribosomal peptide synthetase (NRPS) called Ces. Although a thiotemplate mechanisms involving cyclo-trimerization of ready-made D-O-Leu-D-Ala-L-O-Val-L-Val via a thioesterase (TE) domain is proposed for Cereulide biosynthesis, the exact mechanism is far from being understood. UPLC-TOF MS analysis of B. cereus strains in combination with 13C-labeling experiments now revealed tetra-, octa-, and dodecapeptides of a different sequence, namely (L-O-Val-L-Val-D-O-Leu-D-Ala)1-3, as intermediates of Cereulide biosynthesis. Surprisingly, also di-, hexa-, and decadepsipeptides were identified which, together with the structures of the previously reported isoCereulides E, F, and G, do not correlate to the currently proposed mechanism for Cereulide biosynthesis and violate the canonical NRPS biosynthetic logic. UPLC-TOF MS metabolite analysis and bioinformatic gene cluster analysis highlighted dipeptides rather than single amino or hydroxy acids as the basic modules in tetradepsipeptide assembly and proposed the CesA C-terminal C* domain and the CesB C-terminal TE domain to function as a cooperative esterification and depsipeptide elongation center repeatedly recruiting the action of the C* domain to oligomerize tetradepsipeptides prior to the release of Cereulide from the TE domain by macrocyclization.

  • Chemodiversity of Cereulide, the emetic toxin of Bacillus cereus
    Analytical and Bioanalytical Chemistry, 2015
    Co-Authors: Sandra Marxen, Elrike Frenzel, Monika Ehling-schulz, Genia Lucking, Siegfried Scherer, Timo D Stark, Andrea Rütschle, Gabriel Pürstinger, Elena E. Pohl, Thomas Hofmann
    Abstract:

    Food-borne intoxications are increasingly caused by the dodecadepsipeptide Cereulide, the emetic toxin produced by Bacillus cereus . As such intoxications pose a health risk to humans, a more detailed understanding on the chemodiversity of this toxin is mandatory for the reliable risk assessment of B. cereus toxins in foods. Mass spectrometric screening now shows a series of at least 18 Cereulide variants, among which the previously unknown isoCereulides A–G were determined for the first time by means of UPLC-TOF MS and ion-trap MS^n sequencing, ^13C-labeling experiments, and post-hydrolytic dipeptide and enantioselective amino acid analysis. The data demonstrate a high microheterogeneity in Cereulide and show evidence for a relaxed proof reading function of the non-ribosomal Cereulide peptide synthetase complex giving rise to an enhanced Cereulide chemodiversity. Most intriguingly, the isoCereulides were found to differ widely in their cell toxicity correlating with their ionophoric properties (e.g., purified isoCereulide A showed about 8-fold higher cytotoxicity than purified Cereulide in the HEp-2 assay and induced an immediate breakdown of bilayer membranes). These findings provide a substantial contribution to the knowledge-based risk assessment of B. cereus toxins in foods, representing a still unsolved challenge in the field of food intoxications. Graphical Abstract Bacillus cereus produces Cereulide and a series of previously unknown isoCereulides showing different cytotoxicity and ionophoric properties

  • chemodiversity of Cereulide the emetic toxin of bacillus cereus
    Analytical and Bioanalytical Chemistry, 2015
    Co-Authors: Sandra Marxen, Elrike Frenzel, Monika Ehlingschulz, Genia Lucking, Siegfried Scherer, Timo D Stark, Andrea Rütschle, Gabriel Pürstinger, Elena E. Pohl, Thomas Hofmann
    Abstract:

    Food-borne intoxications are increasingly caused by the dodecadepsipeptide Cereulide, the emetic toxin produced by Bacillus cereus. As such intoxications pose a health risk to humans, a more detailed understanding on the chemodiversity of this toxin is mandatory for the reliable risk assessment of B. cereus toxins in foods. Mass spectrometric screening now shows a series of at least 18 Cereulide variants, among which the previously unknown isoCereulides A–G were determined for the first time by means of UPLC-TOF MS and ion-trap MSn sequencing, 13C-labeling experiments, and post-hydrolytic dipeptide and enantioselective amino acid analysis. The data demonstrate a high microheterogeneity in Cereulide and show evidence for a relaxed proof reading function of the non-ribosomal Cereulide peptide synthetase complex giving rise to an enhanced Cereulide chemodiversity. Most intriguingly, the isoCereulides were found to differ widely in their cell toxicity correlating with their ionophoric properties (e.g., purified isoCereulide A showed about 8-fold higher cytotoxicity than purified Cereulide in the HEp-2 assay and induced an immediate breakdown of bilayer membranes). These findings provide a substantial contribution to the knowledge-based risk assessment of B. cereus toxins in foods, representing a still unsolved challenge in the field of food intoxications.

Thomas Hofmann - One of the best experts on this subject based on the ideXlab platform.

  • first insights into within host translocation of the bacillus cereus toxin Cereulide using a porcine model
    Frontiers in Microbiology, 2018
    Co-Authors: Tobias Bauer, Wolfgang Sipos, Timo D Stark, Tobias Kaser, Christian Knecht, Rene Brunthaler, Armin Saalmuller, Thomas Hofmann, Monika Ehlingschulz
    Abstract:

    Bacillus cereus is a gram-positive pathogen mainly known to evoke two types of foodborne poisonings. The diarrheal syndrome is caused by enterotoxins produced during growth in the intestine. In contrast, the emetic type is caused by the dodecadepsipeptide Cereulide pre-formed in food. Usually, both diseases are self-limiting but occasionally more severe forms, including fatal ones, are reported. Since the mechanisms of Cereulide toxin uptake and translocation within the body as well as the mechanism of its toxic action are still unknown, we used a porcine model to investigate the uptake, routes of excretion and distribution of Cereulide within the host. Pigs were orally challenged with Cereulide using single doses of 10 to 150 µg Cereulide kg-1 body weight to study acute effects or using daily doses of 10 µg Cereulide kg-1 body weight administered for 7 days to investigate effects of longtime, chronic expose. Our study showed that part of Cereulide ingested with food is rapidly excreted with feces while part of the Cereulide toxin is absorbed, passes through membranes and is distributed within the body. Results from the chronic trial indicate bioaccumulation of Cereulide in certain tissues and organs, such as kidney, liver, muscles and fat tissues. Beside its detection in various tissues and organs, our study also demonstrated that Cereulide is able to cross the blood-brain-barrier, which may partially explain the cerebral effects reported from human intoxication cases. The neurobehavioral symptoms, such as seizures and lethargy, observed in our porcine model resemble those reported from human food borne intoxications. The rapid onset of these symptoms indicates direct effects of Cereulide on the central nervous system (CNS), which warrant further research. The porcine model presented here might be useful to study the specific neurobiological effect in detail. Furthermore, our study revealed that typical diagnostic specimens used in human medicine, such as blood samples and urine, are not suitable for diagnostics of food borne Cereulide intoxications. Instead, screening of fecal samples by SIDA-LC-MS may represent a simple and non-invasive method for detection of Cereulide intoxications in clinical settings as well as in foodborne outbreak situations.

  • ces locus embedded proteins control the non ribosomal synthesis of the Cereulide toxin in emetic bacillus cereus on multiple levels
    Frontiers in Microbiology, 2015
    Co-Authors: Genia Lucking, Elrike Frenzel, Siegfried Scherer, Timo D Stark, Thomas Hofmann, Sandra Marxen, Andrea Rütschle, Monika Ehlingschulz
    Abstract:

    The emetic toxin Cereulide produced by Bacillus cereus is synthesized by the modular enzyme complex Ces that is encoded on a pXO1-like megaplasmid. To decipher the role of the genes adjacent to the structural genes cesA/cesB, coding for the non-ribosomal peptide synthetase (NRPS), gene inactivation- and overexpression mutants of the emetic strain F4810/72 were constructed and their impact on Cereulide biosynthesis was assessed. The hydrolase CesH turned out to be a part of the complex regulatory network controlling Cereulide synthesis on a transcriptional level, while the ABC transporter CesCD was found to be essential for post-translational control of Cereulide synthesis. Using a gene inactivation approach, we show that the NRPS activating function of the phosphopantetheinyl transferase (PPtase) embedded in the ces locus was complemented by a chromosomally encoded Sfp-like PPtase, representing an interesting example for the functional interaction between a plasmid encoded NRPS and a chromosomally encoded activation enzyme. In summary, our results highlight the complexity of Cereulide biosynthesis and reveal multiple levels of toxin formation control. ces operon internal genes were shown to play a pivotal role by acting at different levels of toxin production, thus complementing the action of the chromosomal key transcriptional regulators AbrB and CodY.

  • multiparametric quantitation of the bacillus cereus toxins Cereulide and isoCereulides a g in foods
    Journal of Agricultural and Food Chemistry, 2015
    Co-Authors: Sandra Marxen, Elrike Frenzel, Monika Ehlingschulz, Genia Lucking, Siegfried Scherer, Timo D Stark, Andrea Rütschle, Thomas Hofmann
    Abstract:

    Consumption of food products contaminated with Cereulide (1), a toxin produced by Bacillus cereus, might cause intoxications with symptoms reported to range from indigestion pain and emesis to death. Recently, a series of structural variants, coined isoCereulides A–G (2–8), were identified for the first time to be produced along with Cereulide (1). The observation that isoCereulide A (2) shows an ∼8-fold increased cytotoxicity when compared to 1 urges the development of analytical tools enabling an accurate quantitation of these toxins. Therefore, a rapid, sensitive, and robust stable isotope dilution assay (SIDA) was developed for the combined quantitation of 1–8 by means of UPLC-MS/MS. On average, trueness and precision of the method were 112.5 ± 1.8% RSD, repeatability and reproducibility were 2 and 4% for Cereulide and isoCereulides A–G, and the LOD and LOQ of 0.1 and 0.5 ng/g, respectively, demonstrated a high sensitivity for the developed SIDA method. Application of this method to food samples revea...

  • depsipeptide intermediates interrogate proposed biosynthesis of Cereulide the emetic toxin of bacillus cereus
    Scientific Reports, 2015
    Co-Authors: Sandra Marxen, Elrike Frenzel, Monika Ehlingschulz, Genia Lucking, Siegfried Scherer, Timo D Stark, Andrea Rütschle, Thomas Hofmann
    Abstract:

    Cereulide and isoCereulides A-G are biosynthesized as emetic toxins by Bacillus cereus via a non-ribosomal peptide synthetase (NRPS) called Ces. Although a thiotemplate mechanisms involving cyclo-trimerization of ready-made D-O-Leu-D-Ala-L-O-Val-L-Val via a thioesterase (TE) domain is proposed for Cereulide biosynthesis, the exact mechanism is far from being understood. UPLC-TOF MS analysis of B. cereus strains in combination with 13C-labeling experiments now revealed tetra-, octa-, and dodecapeptides of a different sequence, namely (L-O-Val-L-Val-D-O-Leu-D-Ala)1-3, as intermediates of Cereulide biosynthesis. Surprisingly, also di-, hexa-, and decadepsipeptides were identified which, together with the structures of the previously reported isoCereulides E, F, and G, do not correlate to the currently proposed mechanism for Cereulide biosynthesis and violate the canonical NRPS biosynthetic logic. UPLC-TOF MS metabolite analysis and bioinformatic gene cluster analysis highlighted dipeptides rather than single amino or hydroxy acids as the basic modules in tetradepsipeptide assembly and proposed the CesA C-terminal C* domain and the CesB C-terminal TE domain to function as a cooperative esterification and depsipeptide elongation center repeatedly recruiting the action of the C* domain to oligomerize tetradepsipeptides prior to the release of Cereulide from the TE domain by macrocyclization.

  • Chemodiversity of Cereulide, the emetic toxin of Bacillus cereus
    Analytical and Bioanalytical Chemistry, 2015
    Co-Authors: Sandra Marxen, Elrike Frenzel, Monika Ehling-schulz, Genia Lucking, Siegfried Scherer, Timo D Stark, Andrea Rütschle, Gabriel Pürstinger, Elena E. Pohl, Thomas Hofmann
    Abstract:

    Food-borne intoxications are increasingly caused by the dodecadepsipeptide Cereulide, the emetic toxin produced by Bacillus cereus . As such intoxications pose a health risk to humans, a more detailed understanding on the chemodiversity of this toxin is mandatory for the reliable risk assessment of B. cereus toxins in foods. Mass spectrometric screening now shows a series of at least 18 Cereulide variants, among which the previously unknown isoCereulides A–G were determined for the first time by means of UPLC-TOF MS and ion-trap MS^n sequencing, ^13C-labeling experiments, and post-hydrolytic dipeptide and enantioselective amino acid analysis. The data demonstrate a high microheterogeneity in Cereulide and show evidence for a relaxed proof reading function of the non-ribosomal Cereulide peptide synthetase complex giving rise to an enhanced Cereulide chemodiversity. Most intriguingly, the isoCereulides were found to differ widely in their cell toxicity correlating with their ionophoric properties (e.g., purified isoCereulide A showed about 8-fold higher cytotoxicity than purified Cereulide in the HEp-2 assay and induced an immediate breakdown of bilayer membranes). These findings provide a substantial contribution to the knowledge-based risk assessment of B. cereus toxins in foods, representing a still unsolved challenge in the field of food intoxications. Graphical Abstract Bacillus cereus produces Cereulide and a series of previously unknown isoCereulides showing different cytotoxicity and ionophoric properties

Michio Ohta - One of the best experts on this subject based on the ideXlab platform.

  • a new rapid and sensitive detection method for Cereulide producing bacillus cereus using a cycleave real time pcr
    Letters in Applied Microbiology, 2009
    Co-Authors: M Yabutani, Norio Agata, Michio Ohta
    Abstract:

    Aims:  To develop a rapid and sensitive detection method for Cereulide-producing Bacillus cereus using a real-time PCR based on the sequence of the Cereulide synthesis gene. Methods and Results:  A total of 56 Cereulide-producing B. cereus and 15 Cereulide-negative strains were tested. We designed specific primers and probes for the detection of Cereulide-producing B. cereus. The new cycleave real-time PCR assay gave positive detections for all of 56 Cereulide-producing B. cereus strains, whereas all other strains including 10 systemic infectious disease strains were negative. No cross-reaction was observed and the internal control showed positive for all samples. Conclusions:  The performance of the assay was highly reproducible and specific for Cereulide-producing B. cereus. The positive detection was obtained within only 2 h for Cereulide-producing strains. The detection limit of this assay was evaluated as 104 CFU g−1 food sample. The assay also confirmed that strains from systemic infectious cases were Cereulide-negative. Significance and Impact of the Study:  This assay is applicable for contaminated foods as well as specimens from infectious disease cases. We recommend this assay for routine examination of suspected B. cereus food poisonings.

  • a new phylogenetic cluster of Cereulide producing bacillus cereus strains
    Journal of Clinical Microbiology, 2007
    Co-Authors: Maria Vassileva, Norio Agata, Tadao Hasegawa, Keizo Torii, Megumi Oshimoto, Akira Okamoto, Keiko Yamada, Michio Ohta
    Abstract:

    Phenotypic and molecular studies have established that Cereulide-producing strains of Bacillus cereus are a distinct and probably recently emerged clone within the Bacillus population. We analyzed a set of B. cereus strains, both Cereulide producers and nonproducers, by multilocus sequence typing. Consistent with earlier reports, nonproducers demonstrated high heterogeneity. Most Cereulide-producing strains and all flagellar antigen type H1 strains were allocated to the known sequence type of exclusively emetic B. cereus strains. Several Cereulide-producing strains, however, were recovered at a new phylogenetic location, all of which were serotype H3 or H12. We hypothesize that the group of Cereulide producers is diversifying progressively, probably by lateral transfer of the corresponding gene complex.

  • quantitative analysis of Cereulide an emetic toxin of bacillus cereus by using rat liver mitochondria
    Microbiology and Immunology, 2005
    Co-Authors: Kumiko Kawamurasato, Norio Agata, Yumi Hirama, Kenzo Torii, Akira Takeno, Tadao Hasegawa, Yoshiharu Shimomura, Michio Ohta
    Abstract:

    An emetic toxin Cereulide, produced by Bacillus cereus, causes emetic food poisonings, but a method for quantitative measurement of Cereulide has not been well established. A current detection method is a bioassay method using the HEp-2 cell vacuolation test, but it was unable to measure an accurate concentration. We established a quantitative assay for Cereulide based on its mitochondrial respiratory uncoupling activity. The oxygen consumption in a reaction medium containing rat liver mitochondria was rapid in the presence of Cereulide. Thus uncoupling effect of Cereulide on mitochondrial respiration was similar to those of uncouplers 2,4-dinitrophenol (DNP), carbonylcyanide m-chlorophenylhydrazone (CCCP), and valinomycin. This method gave constant results over a wide range of Cereulide concentrations, ranging from 0.05 to 100 μg/ml. The minimum Cereulide concentration to detect uncoupled oxygen consumption was 50 ng/ml and increased dose-dependently to the maximum level. Semi-log relationship between the oxygen consumption rate and the Cereulide concentraiton enables this method to quantify Cereulide. The results of this method were highly reproducible as compared with the HEp-2 cell vacuolation test and were in good agreement with those of the HEp-2 cell vacuolation test. The enterotoxin of B. cereus or Staphylococcus aureus did not show any effect on the oxygen consumption, indicating this method is specific for the identification of Cereulide as a causative agent of emetic food poisonings.

  • molecular diversity of Cereulide detected by means of nano hplc esi q tof ms
    International Journal of Mass Spectrometry, 2004
    Co-Authors: Suthasinee Pitchayawasin, Minoru Isobe, Norio Agata, Masaki Kuse, Thomas Franz, Michio Ohta
    Abstract:

    Abstract Cereulide is a cyclic dodecadepsipeptide from a pathogenic bacteria Bacillus cereus , which shows the emetic toxicity. Molecular diversity, or variety in homologation was found as a minor constituent of this cyclic peptide. Its molecular weight is 1152 but its homologs were observed as 1138 and 1166, which had 14 mass lower and higher differences from Cereulide. This homologation was observed in about 10% of Cereulide. It seemed to be difficult to determine the heterogeneous amino acids directly by MS/MS analysis on the intact molecules of Cereulide. And hydrolysis of this cyclic peptide gave dipeptides, which were analyzed to determine their heterogeneous components by means of nano-HPLC-ESI-Q-TOF-MS and MS/MS. Among all amino- and oxy-acids, we found that O -Val and O -Leu were the keys of variation in Cereulide. These findings will be significant to establish an identification method for pathogenic bacteria on the basis of biosynthetic pathways.

  • synthase de Cereulide produite par bacillus cereus gene codant pour celle ci et procede de detection de Cereulide
    2003
    Co-Authors: Michio Ohta, Norio Agata
    Abstract:

    L'invention concerne un procede permettant de detecter rapidement et de maniere adequate le Cereulide, une toxine emetique produite par Bacillus cereus. On detecte le Cereulide dans un echantillon grâce a la presence de Cereulide synthase, qui sert d'indicateur. La presence de Cereulide synthase est examinee par la detection d'un acide nucleique codant pour cette enzyme, ou par un procede immunologique utilisant un anticorps specifique de l'enzyme.

Minoru Isobe - One of the best experts on this subject based on the ideXlab platform.

  • in vitro antimicrobial activity of Cereulide and valinomycin compare with cyclic d l α peptides and their enhanced effect
    International journal of pharma and bio sciences, 2015
    Co-Authors: Arthit Makarasen, Nanthawan Reukngam, Piyachat Chuysinuan, Minoru Isobe, Nitirat Chimnoi, Supanna Techasakul
    Abstract:

    Cereulide and valinomycin are both cyclic depsipeptides with 12 stereogenic centers that have a very similar sequence in the structure. Both compounds are hexagonal cylinder-like framework and all the side chains stick outside of the framework. The framework of cyclic D,L-α-peptides were structured different from the Cereulide and valinomycin which are performed by cyclic DDLL-peptides. The cyclic D,L-α-peptides consisted of an even number of alternating D- and L- α-amino acid which can adopt flat and stack to form hollow, β-sheet like tubular structures. The amino acid side chains are presented on the outside surface of the framework and the interior surface of such a tube is hydrophilic. In our studies, we found that the original Cereulide and valinomycin demonstrated higher antimicrobial activity than the cyclic D,L-α-peptides which are synthesized by using Cereulide and valinomycin as a model structure. Furthermore, the combinations of Cereulide or valinomycin with clinically used drugs against fungi were found to enhance the antimicrobial efficacy by decreasing the MIC values between 2-128 times.

  • synthesis of four lysine linkedCereulide analogues showing ionophoric activity towards potassiumcations as lead compounds for emetic toxin detection by immunoassays
    Synthesis, 2009
    Co-Authors: Arthit Makarasen, Toshio Nishikawa, Minoru Isobe
    Abstract:

    An improved total synthesis of the emetic toxin Cereulide and the preparation of four lysine-linked Cereulide analogues is described. The Cereulide analogues are prepared by replacing one of the amino acid residues in Cereulide with lysine. The Cereulide analogues demonstrate ionophoric activity towards alkali metal ions and inorganic ammonium ions, and are currently being used to develop an enzyme-linked immunosorbent assay for Cereulide.

  • higher structure of Cereulide an emetic toxin from bacillus cereus and special comparison with valinomycin an antibiotic from streptomyces fulvissimus
    Chemistry-an Asian Journal, 2009
    Co-Authors: Arthit Makarasen, Minoru Isobe, Kenji Yoza
    Abstract:

    : Cereulide and valinomycin are both 36-membered cyclic depsipeptides with 12 stereogenic centers that have a very similar sequence of cyclo [-D-O-Leu-D-Ala-L-O-Val-L-Val-]3 and cyclo [-D-O-Val-D-Val-L-O-Ala-L-Val-]3, respectively. Cereulide is an emetic toxin produced by Bacillus cereus through an unusual non-ribosomal peptide synthesis (NRPS), whereas valinomycin, produced by Streptomyces fulvissimus, is a known antibiotic drug. Both compounds are known as K+-ion-selective ionophores and cause a potassium-dependent drop in the transmembrane potential of mitochondria, arising from the uptake of a K+-ion-charged ionophore complex. Such compounds may affect mitochondrial function. In the three-dimensional structure of Cereulide and valinomycin, Cereulide has a vertical and horizontal mirror-image-like structure as is the case in valinomycin. The only difference is the side chains which are linked to a similar framework. Through the current 1H NMR spectroscopy and metal-complexation studies, we found that Cereulide had a higher complexation ability to metal ions compared to valinomycin. Cereulide exhibited the K+-ion-selective ionophore property at a lower concentration than valinomycin. X-ray crystallographic analyses of the Cereulide and valinomycin H+ form were compared, and revealed that the higher structures of both compounds also showed similarity in the crystal structures. The structure of Cereulide-H+ form was found to be in agreement with the structure obtained by a combination of NMR spectroscopy and molecular-mechanics calculations, which afforded reasonable dihedral angles at the local-minimum-energy conformation of the Cereulide-K+-ion complex.

  • molecular diversity of Cereulide detected by means of nano hplc esi q tof ms
    International Journal of Mass Spectrometry, 2004
    Co-Authors: Suthasinee Pitchayawasin, Minoru Isobe, Norio Agata, Masaki Kuse, Thomas Franz, Michio Ohta
    Abstract:

    Abstract Cereulide is a cyclic dodecadepsipeptide from a pathogenic bacteria Bacillus cereus , which shows the emetic toxicity. Molecular diversity, or variety in homologation was found as a minor constituent of this cyclic peptide. Its molecular weight is 1152 but its homologs were observed as 1138 and 1166, which had 14 mass lower and higher differences from Cereulide. This homologation was observed in about 10% of Cereulide. It seemed to be difficult to determine the heterogeneous amino acids directly by MS/MS analysis on the intact molecules of Cereulide. And hydrolysis of this cyclic peptide gave dipeptides, which were analyzed to determine their heterogeneous components by means of nano-HPLC-ESI-Q-TOF-MS and MS/MS. Among all amino- and oxy-acids, we found that O -Val and O -Leu were the keys of variation in Cereulide. These findings will be significant to establish an identification method for pathogenic bacteria on the basis of biosynthetic pathways.

  • high incorporation of l amino acids to Cereulide an emetic toxin from bacillus cereus
    Bioorganic & Medicinal Chemistry Letters, 2000
    Co-Authors: Masaki Kuse, Minoru Isobe, Norio Agata, Thomas Franz, Kazushi Koga, Sathorn Suwan, Michio Ohta
    Abstract:

    Abstract Cereulide is a principal toxin causing emetic syndrome produced by Bacillus cereus . This paper deals with biosynthetic studies on this unusual cyclic depsipeptide toxin from 13 C labeled l -amino acid precursors (Val, Leu, Ala) upon cultivation in synthetic media. The analyses were made at atomic level of the constituent amino- or oxy-acids through NMR and ESI-MS/MS spectroscopic methods on Cereulide and its hydrolysate dipeptides. The incorporation of the 13 C atom was 95% in each O -Val, O -Leu and l -Val, while 40% in d -Ala of Cereulide.