Impact of Phytochemicals on Viability and Cereulide Toxin Synthesis in Bacillus cereus Revealed by a Novel High-Throughput Method, Coupling an AlamarBlue-Based Assay with UPLC-MS/MS.

Due to its food-poisoning potential, Bacillus cereus has attracted the attention of the food industry. The cereulide-toxin-producing subgroup is of particular concern, as cereulide toxin is implicated in broadscale food-borne outbreaks and occasionally causes fatalities. The health risks associated with long-term cereulide exposure at low doses remain largely unexplored. Natural substances, such as plant-based secondary metabolites, are widely known for their effective antibacterial potential, which makes them promising as ingredients in food and also as a surrogate for antibiotics. In this work, we tested a range of structurally related phytochemicals, including benzene derivatives, monoterpenes, hydroxycinnamic acid derivatives and vitamins, for their inhibitory effects on the growth of B. cereus and the production of cereulide toxin. For this purpose, we developed a high-throughput, small-scale method which allowed us to analyze B. cereus survival and cereulide production simultaneously in one workflow by coupling an AlamarBlue-based viability assay with ultraperformance liquid chromatography-mass spectrometry (UPLC-MS/MS). This combinatory method allowed us to identify not only phytochemicals with high antibacterial potential, but also ones specifically eradicating cereulide biosynthesis already at very low concentrations, such as gingerol and curcumin.

Characterization of Bacillus cereus group isolates from powdered food products.

Mashed potato powder as well as powdered infant formula (PIF) are frequently contaminated with Bacillus cereus sensu lato (B. cereus s.l.), mainly with its spores. These products have also been implicated in foodborne illnesses. Here, we characterized B. cereus s.l. isolates originating from powdered products based on sporulation assays, toxin gene profiling, and panC typing combined with a SplitsTree analysis. Furthermore, cytotoxicity assays with B. cytotoxicus isolates were performed. 78% of PIF tested positive for B. cereus s.l., whereas 92% of all mashed potato powders were positive. In total, 43 isolates were further characterized. The nhe and cytK2 genes were most frequently detected. Moreover, a cereulide-producer was detected from PIF. Most isolates were assigned to panC group III, but members of group II, IV, V, and VII could also be found. Nine B. cytotoxicus were isolated out of nine mashed potato powders. All panC group VII isolates were positive for cytK1. Cytotoxicity assays of these nine isolates revealed one highly cytotoxic strain, while all other isolates exhibited no detectable cytotoxicity, underpinning that cytotoxicity of a certain B. cereus group strain cannot be deduced from the sole presence or absence of toxin genes.

Two New Cyclic Depsipeptides from the Endophytic Fungus Fusarium sp.

Two new cyclic depsipeptides, W493 C (1) and D (2), along with two known derivatives W493 A (3) and B (4) were obtained from the endophytic fungus Fusarium sp. isolated from the Mangrove plant Ceriops tagal. The structures of the new compounds were determined on the basis of one- and two dimensional NMR and high-resolution mass spectroscopic data. The absolute configurations of the amino acid residues of 1 and 2 were confirmed by application of Marfey's method. W493 A (3) and B (4) exhibited moderate activity against the fungus Cladosporium cladosporiodes and weak antitumor activity against the human ovarian cancer cell line A2780.

Effects of sugar and amino acid supplementation on Aureobasidium pullulans NRRL 58536 antifungal activity against four Aspergillus species.

Cultured cell extracts from ten tropical strains of Aureobasidium pullulans were screened for antifungal activity against four pathogenic Aspergillus species (Aspergillus flavus, Aspergillus niger, Aspergillus fumigatus, and Aspergillus terreus) using the well diffusion and conidial germination inhibition assays. The crude cell extract from A. pullulans NRRL 58536 resulted in the greatest fungicidal activity against all four Aspergillus species and so was selected for further investigation into enhancing the production of antifungal activity through optimization of the culture medium, carbon source (sucrose and glucose) and amino acid (phenylalanine, proline, and leucine) supplementation. Sucrose did not support the production of any detectable antifungal activity, while glucose did with the greatest antifungal activity against all four Aspergillus species being produced in cells grown in medium containing 2.5 % (w/v) glucose. With respect to the amino acid supplements, variable trends between the different Aspergillus species and amino acid combinations were observed, with the greatest antifungal activities being obtained when grown with phenylalanine plus leucine supplementation for activity against A. flavus, proline plus leucine for A. terreus, and phenylalanine plus proline and leucine for A. niger and A. fumigatus. Thin layer chromatography, spectrophotometry, high-performance liquid chromatography, (1)H-nuclear magnetic resonance, and MALDI-TOF mass spectrometry analyses were all consistent with the main component of the A. pullulans NRRL 58536 extracts being aureobasidins.

Ciclohexadespipeptide beauvericin degradation by different strains of Saccharomyces cerevisiae.

The interaction between the mycotoxin beauvericin (BEA) and 9 yeast strains of Saccharomyces cerevisiae named LO9, YE-2, YE5, YE-6, YE-4, A34, A17, A42 and A08 was studied. The biological degradations were carried out under aerobic conditions in the liquid medium of Potato Dextrose Broth (PDB) at 25°C for 48 h and in a food/feed system composed of corn flour at 37°C for 3 days, respectively. BEA present in fermented medium and corn flour was determined using liquid chromatography coupled to the mass spectrometry detector in tandem (LC-MS/MS) and the BEA degradation products produced during the fermentations were determined using the technique of the liquid chromatography coupled to a linear ion trap (LIT). Results showed that the S. cerevisiae strains reduced meanly the concentration of the BEA present in PDB by 86.2% and in a food system by 71.1%. All the S. cerevisiae strains used in this study showed a significant BEA reduction during the fermentation process employed.

Potato crop as a source of emetic Bacillus cereus and cereulide-induced mammalian cell toxicity.

Bacillus cereus, aseptically isolated from potato tubers, were screened for cereulide production and for toxicity on human and other mammalian cells. The cereulide-producing isolates grew slowly, the colonies remained small (~1 mm), tested negative for starch hydrolysis, and varied in productivity from 1 to 100 ng of cereulide mg (wet weight)(-1) (~0.01 to 1 ng per 10(5) CFU). By DNA-fingerprint analysis, the isolates matched B. cereus F5881/94, connected to human food-borne illness, but were distinct from cereulide-producing endophytes of spruce tree (Picea abies). Exposure to cell extracts (1 to 10 µg of bacterial biomass ml(-1)) and to purified cereulide (0.4 to 7 ng ml(-1)) from the potato isolates caused mitochondrial depolarization (loss of ΔΨm) in human peripheral blood mononuclear cells (PBMC) and keratinocytes (HaCaT), porcine spermatozoa and kidney tubular epithelial cells (PK-15), murine fibroblasts (L-929), and pancreatic insulin-producing cells (MIN-6). Cereulide (10 to 20 ng ml(-1)) exposed pancreatic islets (MIN-6) disintegrated into small pyknotic cells, followed by necrotic death. Necrotic death in other test cells was observed only after a 2-log-higher exposure. Exposure to 30 to 60 ng of cereulide ml(-1) induced K(+) translocation in intact, live PBMC, keratinocytes, and sperm cells within seconds of exposure, depleting 2 to 10% of the cellular K(+) stores within 10 min. The ability of cereulide to transfer K(+) ions across biological membranes may benefit the producer bacterium in K(+)-deficient environments such as extracellular spaces inside plant tissue but is a pathogenic trait when in contact with mammalian cells.

Metabolism as a key to histone deacetylase inhibition.

There is growing interest in the epigenetic mechanisms that are dysregulated in cancer and other human pathologies. Under this broad umbrella, modulators of histone deacetylase (HDAC) activity have gained interest as both cancer chemopreventive and therapeutic agents. Of the first generation, FDA-approved HDAC inhibitors to have progressed to clinical trials, vorinostat represents a "direct acting" compound with structural features suitable for docking into the HDAC pocket, whereas romidepsin can be considered a prodrug that undergoes reductive metabolism to generate the active intermediate (a zinc-binding thiol). It is now evident that other agents, including those in the human diet, can be converted by metabolism to intermediates that affect HDAC activity. Examples are cited of short-chain fatty acids, seleno-α-keto acids, small molecule thiols, mercapturic acid metabolites, indoles, and polyphenols. The findings are discussed in the context of putative endogenous HDAC inhibitors generated by intermediary metabolism (e.g. pyruvate), the yin-yang of HDAC inhibition versus HDAC activation, and the screening assays that might be most appropriate for discovery of novel HDAC inhibitors in the future.

Phytotoxins, elicitors and other secondary metabolites from phytopathogenic "blackleg" fungi: structure, phytotoxicity and biosynthesis.

The metabolites produced by the fungal species Leptosphaeria maculans and L. biglobosa under different culture conditions, together with their phytotoxic activities are reviewed. In addition, the biosynthetic studies of blackleg metabolites carried out to date are described and suggestions for species reclassification are provided.