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Emerging Fusarium and Alternaria mycotoxins gain more and more interest due to their frequent contamination of food and feed, although in vivo toxicity and toxicokinetic data are limited. Whereas the Fusarium mycotoxins beauvericin, moniliformin and enniatins particularly contaminate grain and grain-based products, Alternaria mycotoxins are also detected in fruits, vegetables and wines. Although contamination levels are usually low (µg/kg range), higher contamination levels of enniatins and tenuazonic acid may occasionally occur. In vitro studies suggest genotoxic effects of enniatins A, A1 and B1, beauvericin, moniliformin, alternariol, alternariol monomethyl ether, altertoxins and stemphyltoxin-III. Furthermore, in vitro studies suggest immunomodulating effects of most emerging toxins and a reproductive health hazard of alternariol, beauvericin and enniatin B. More in vivo toxicity data on the individual and combined effects of these contaminants on reproductive and immune system in both humans and animals is needed to update the risk evaluation by the European Food Safety Authority. Taking into account new occurrence data for tenuazonic acid, the complete oral bioavailability, the low total body clearance in pigs and broiler chickens and the limited toxicity data, a health risk cannot be completely excluded. Besides, some less known Alternaria toxins, especially the genotoxic altertoxins and stemphyltoxin III, should be incorporated in risk evaluation as well.

Staphylococci secrete autoinducing peptides (AIPs) as signalling molecules to regulate population-wide behaviour. AIPs from non- Staphylococcus aureus staphylococci have received attention as potential antivirulence agents to inhibit quorum sensing and virulence gene expression in the human pathogen Staphylococcus aureus . However, only a limited number of AIP structures from non- S. aureus staphylococci have been identified to date, as the minute amounts secreted in complex media render it difficult. Here, we report a method for the identification of AIPs by exploiting their thiolactone functionality for chemoselective trapping and enrichment of the compounds from the bacterial supernatant. Standard liquid chromatography mass spectrometry analysis, guided by genome sequencing data, then readily provides the AIP identities. Using this approach, we confirm the identity of five known AIPs and identify the AIPs of eleven non- S. aureus species, and we expect that the method should be extendable to AIP-expressing Gram-positive bacteria beyond the Staphylococcus genus. Bacteria communicate through excretion of minute amounts of chemical signalling molecules that affect virulence, biofilm formation and colonization. In staphylococci, these molecules, called autoinducing peptides, are macrocyclic thiolactone-containing peptides. Now, a simple enrichment method, based on chemoselective capture on polymer beads, has been developed that enables the identification of previously unknown autoinducing peptides.

Destruxins (DTXs) are cyclic depsipeptides produced by many Metarhizium isolates that have long been assumed to contribute to virulence of these entomopathogenic fungi. We evaluated the virulence of 20 Metarhizium isolates against insect larvae and measured the concentration of DTXs A, B, and E produced by these same isolates in submerged (shaken) cultures. Eight of the isolates (ARSEF 324, 724, 760, 1448, 1882, 1883, 3479, and 3918) did not produce DTXs A, B, or E during the five days of submerged culture. DTXs were first detected in culture medium at 2-3 days in submerged culture. Galleria mellonella and Tenebrio molitor showed considerable variation in their susceptibility to the Metarhizium isolates. The concentration of DTXs produced in vitro did not correlate with percent or speed of insect kill. We established endophytic associations of M. robertsii and M. acridum isolates in Vigna unguiculata (cowpeas) and Cucumis sativus (cucumber) plants. DTXs were detected in cowpeas colonized by M. robertsii ARSEF 2575 12 days after fungal inoculation, but DTXs were not detected in cucumber. This is the first instance of DTXs detected in plants endophytically colonized by M. robertsii. This finding has implications for new approaches to fungus-based biological control of pest arthropods.

Cereulide-producing Bacillus cereus, which causes foodborne illnesses with vomiting, and psychrotolerant B. cereus group strains such as Bacillus mycoides, which can grow at ≥7°C and cause spoilage of refrigerated foods, are significant concerns for the food industry. Rapid and simple methods to discriminate the cereulide-producing B. cereus and psychrotolerant B. cereus group strains from other B. cereus group strains are needed. We developed a novel, rapid, and simple method with matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) analysis for simultaneous discrimination of these two groups from other B. cereus group strains. A potassium adduct of cereulide was used to detect cereulide-producing B. cereus, and three ribosomal subunit proteins (L30, S16, and S20) were used to detect psychrotolerant B. cereus group. A total of 51 B. cereus group strains were analyzed by MALDI-TOF MS. The biomarkers allowed successful discrimination of 16 cereulide-producing B. cereus and 15 psychrotolerant B. cereus group strains from other B. cereus group strains. The results showed that this MALDI-TOF MS analysis allows simultaneous discrimination of cereulide-producing B. cereus and psychrotolerant B. cereus group strains from other B. cereus group strains. This efficient method has the potential to be a valuable tool for ensuring food safety.

Social insects are frequently observed in symbiotic association with bacteria that produce antimicrobial natural products as a defense mechanism. There is a lack of studies on the microbiota associated with stingless bees and their antimicrobial compounds. To the best of our knowledge, this study is the first to report the isolation of Paenibacillus polymyxa ALLI-03-01 from the larval food of the stingless bee Melipona scutellaris. The bacterial strain was cultured under different conditions and produced (L)-(−)-3-phenyllactic acid and fusaricidins, which were active against entomopathogenic fungi and Paenibacillus larvae. Our results indicate that such natural products could be related to colony protection, suggesting a defense symbiosis between P. polymyxa ALLI-03-01 and Melipona scutellaris.

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 13 C-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.

Paenibacillus polymyxa-M1 is a potent producer of bioactive compounds, such as lipopeptides, polyketides, and lantibiotics of biotechnological and medical interest. Genome sequencing revealed nine gene clusters for nonribosomal biosynthesis of such agents. Here we report on the investigation of the fusaricidins, a complex of cyclic lipopeptides containing 15-guanidino-3-hydroxypentadecanoic acid (GHPD) as fatty acid component by matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). More than 20 variants of these compounds were detected and characterized in detail. Mass spectrometric sequence analysis was performed by MALDI-LIFT-TOF/TOF fragment analysis. The obtained product ion spectra show a specific processing in the fatty acid part. GHPD is cleaved between the α- and ß-position yielding two fragments a and b, one bearing the end-standing guanidine group and another one comprising the residual two C-atoms of GHPD with the attached peptide moiety. The complete sequence of all fusaricidins was derived from sets of b n - and y n -ions. The fusaricidin complex can be divided into four lipopeptide families, three of them showing variations of the amino acid in position 3, Val or Ile for the first and Tyr or Phe for families 2 and 3, respectively. A collection of novel fusaricidins was detected differing from those of families 1–3 by an additional residue of 71 Da (family 4). LIFT-TOF/TOF fragment spectra of these species imply that in their peptide moiety, an Ala-residue is attached by an ester bond to the free hydroxyl group of Thr 4 . More than 10 novel fusaricidins were characterized mass spectrometrically. Graphical Abstract ᅟ

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, 13 C-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

Fusarium wilt, caused by the fungal pathogen Fusarium oxysporum f. sp. cubense (Foc), is one of the most destructive diseases of banana. Toxins produced by Foc have been proposed to play an important role during the pathogenic process. The objectives of this study were to investigate the contamination of banana with toxins produced by Foc, and to elucidate their role in pathogenesis. Twenty isolates of Foc representing races 1 and 4 were isolated from diseased bananas in five Chinese provinces. Two toxins were consistently associated with Foc, fusaric acid (FA) and beauvericin (BEA). Cytotoxicity of the two toxins on banana protoplast was determined using the Alamar Blue assay. The virulence of 20 Foc isolates was further tested by inoculating tissue culture banana plantlets, and the contents of toxins determined in banana roots, pseudostems and leaves. Virulence of Foc isolates correlated well with toxin deposition in the host plant. To determine the natural occurrence of the two toxins in banana plants with Fusarium wilt symptoms, samples were collected before harvest from the pseudostems, fruit and leaves from 10 Pisang Awak 'Guangfen #1' and 10 Cavendish 'Brazilian' plants. Fusaric acid and BEA were detected in all the tissues, including the fruits. The current study provides the first investigation of toxins produced by Foc in banana. The toxins produced by Foc, and their levels of contamination of banana fruits, however, were too low to be of concern to human and animal health. Rather, these toxins appear to contribute to the pathogenicity of the fungus during infection of banana plants.

Yogurt, a milk-derived product, is susceptible to mycotoxin contamination. While various methods have been developed for the analysis of dairy products, only a few have been specifically validated for yogurt. In addition, these methods are primarily focus on detecting aflatoxins and zearalenone. This study aimed to conduct a preliminary investigation into the presence of regulated, emerging, and modified mycotoxins in natural and oat yogurts available in the Spanish market. For this, a QuEChERS-based extraction method was optimized and then validated to detect and quantify 32 mycotoxins using ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). The method was in-house validated for the analysis of natural and oat yogurt in terms of linearity, matrix effect, sensitivity, accuracy, and precision. Satisfactory performance characteristics were achieved; for most of the analytes, LOQs were lower than 2 ng/g, and recoveries ranged from 60 to 110% with a precision, expressed as the relative standard deviation of the recovery, lower than 15%. Subsequently, the validated method was applied to analyze commercial yogurt samples, revealing a notable incidence of beauvericin and enniatins, with some analogues found in up to 100% of the samples. Alternariol methyl ether was also frequently found, appearing in 50% of the samples. Additionally, the study identified regulated toxins such as fumonisins, ochratoxin A , and HT-2 toxin. These results provide new incidence data in yogurt, raising concerns about potential health risks for consumers.