Simultaneous Discrimination of Cereulide-Producing Bacillus cereus and Psychrotolerant B. cereus Group by Matrix-Assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometry.

ABSTRACT: 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.

Application of Wastewater-Based Epidemiology for Tracking Human Exposure to Deoxynivalenol and Enniatins.

Wastewater-based epidemiology (WBE) is a promising biomonitoring approach with the potential to provide direct information on human intake and exposure to food contaminants and environmental chemicals. The aim of this study was to apply WBE while employing the normalization method for exploring human exposure to selected mycotoxins according to population biomarker 5-hydroxyindoleacetic acid (5-HIAA). This type of normalization technique has been previously used to detect various other compounds. However, to the best of our knowledge, this is the first study tracking human exposure to mycotoxins. A sensitive analytical methodology was developed to achieve reliable quantification of deoxynivalenol, enniatins, and beauvericin in wastewater (WW) samples. The applicability of the method was evaluated by testing 29 WW samples collected at WW treatment plants in Latvia. With frequency of detection greater than 86%, enniatins B, B1, A, and A1 were revealed in WW samples. The estimated total daily intake for enniatins was in the range of 1.8-27.6 µg/day per person. Free deoxynivalenol (DON) was determined in all analysed WW samples. Based on the average 5-HIAA excretion level and the determined 5-HIAA content in the samples, the intake of DON by the human population of Riga was estimated at 325 ng/kg b.w. day.

Analysis of Mycotoxin and Secondary Metabolites in Commercial and Traditional Slovak Cheese Samples.

Cheese represents a dairy product extremely inclined to fungal growth and mycotoxin production. The growth of fungi belonging to Aspergillus, Penicillium, Fusarium, Claviceps, Alternaria, and Trichoderma genera in or on cheese leads to undesirable changes able to affect the quality of the final products. In the present investigation, a total of 68 types of commercial and traditional Slovak cheeses were analyzed to investigate the occurrence of fungal metabolites. Altogether, 13 fungal metabolites were identified and quantified. Aflatoxin M1, the only mycotoxin regulated in milk and dairy products, was not detected in any case. However, the presence of metabolites that have never been reported in cheeses, such as tryptophol at a maximum concentration level from 13.4 to 7930 µg/kg (average: 490 µg/kg), was recorded. Out of all detected metabolites, enniatin B represents the most frequently detected mycotoxin (0.06-0.71 µg/kg) in the analyzed samples. Attention is drawn to the lack of data on mycotoxins' origin from Slovak cheeses; in fact, this is the first reported investigation. Our results indicate the presence of fungal mycotoxin contamination for which maximum permissible levels are not established, highlighting the importance of monitoring the source and producers of contamination in order to protect consumers' health.

[Simultaneous determination of beauvericin and four enniatins in eggs by ultra-performance liquid chromatography-tandem mass spectrometry coupled with cold-induced liquid-liquid extraction and dispersive solid phase extraction].

Enniatins (ENNs) and beauvericin (BEA), known as emerging mycotoxins, are the toxic secondary metabolites produced by various Fusarium species. Most grain and grain-based products are contaminated with ENNs and BEA. Animals have been exposed to ENNs and BEA primarily due to consumption of cereal grains and cereal by-products. ENNs and BEA have been detected in animal-derived food and human breast milk, and they pose significant threats to public health. Therefore, more contamination data are urgently needed for the risk assessment of ENNs and BEA present in animal-derived food. To ensure the quality of animal-derived food, a method has been developed for the simultaneous detection of five emerging mycotoxins (viz. enniatin B, enniatin B1, enniatin A, enniatin A1, and beauvericin) in eggs by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) coupled with cold-induced liquid-liquid extraction (CI-LLE) and dispersive solid phase extraction (DSPE). The main factors governing the response, recovery, and sensitivity of the method, such as the type of extraction solvent, the temperature and duration of cold treatment in CI-LLE, the type and dosages of adsorbents, and apparatus conditions and the type of mobile phase used, were optimized during sample pretreatment and instrument analysis. The mycotoxin residues in eggs were extracted using 20 mL acetonitrile-water-acetic acid (79∶20∶1, v/v/v) mixture for 20 min by the vortex shock method. After mixing, the mixture was frozen for 30 min in a freezer at -40 ℃ and centrifuged for 10 min at 10000 r/min. A 2 mL aliquot of the upper acetonitrile layer was purified by using 70 mg of C18 adsorbents. After whirling, the mixtures were centrifuged at 10000 r/min for 5 min. The purified solution was then concentrated to nearly dry in nitrogen atmosphere at 40 ℃. The residues were dissolved in 1.0 mL 80%(v/v) acetonitrile aqueous solution. The target analytes were separated on an ACQUITY UPLC BEH C18 chromatographic column (100 mm×2.1 mm, 1.7 µm) at a column temperature of 40 ℃, with a flow rate of 0.3 mL/min. The injection volume was 5 µL, and gradient elution was conducted using acetonitrile and 5 mmol/L ammonium formate solution as the mobile phases. Multiple reactions monitoring (MRM) was conducted in the positive electrospray ionization (ESI +) mode. The isotope internal standard method was used for quantification of BEA, and the matrix-matched external standard method was used for quantification of four ENNs. The results of the optimized method showed that the five analytes were completely separated by using the above-mentioned chromatographic column. Good linear relationships were obtained for the five mycotoxins in the concentration range of 0.1-50.0 µg/L; the correlation coefficient (r2) ranged from 0.9983 to 0.9997. The limits of detection (LODs) ranged from 0.05 to 0.15 µg/kg, while the limits of quantification (LOQs) ranged from 0.20 to 0.50 µg/kg. Accuracy and precision experiments were conducted by spiking egg samples with known amounts of analytes at three concentration levels (0.5, 5.0, and 25.0 µg/kg, in compliance with the current legislation) with six replicates. The average recoveries of the five analytes ranged from 81.1% to 106%, and the relative standard deviations (RSDs) were between 0.27% and 9.79%. The matrix effects of the analytes were between 2.70% and 45.1% in egg samples after pretreatment by CI-LLE coupled with DSPE. The developed method was applied to the determination of five mycotoxins in rural eggs and commercial eggs. BEA was detected in most rural egg samples, with detection rates of 30.4%. None of the four ENN residues were detected. Therefore, we can conclude that the method described herein has the advantages of sensitivity, stabilization, accuracy, good recovery, and easy operation, and is suitable for the simultaneous and rapid determination of BEA and ENN residues in eggs.

Distribution of the Emetic Toxin Cereulide in Cow Milk.

Bacillus cereus is frequently associated with food-borne intoxications, and its emetic toxin cereulide causes emesis and nausea after consumption of contaminated foods. The major source for contamination is found within contaminated raw materials containing the highly chemically resistant cereulide, independent of vegetative bacteria cells. Up to date, non-existing removal strategies for cereulide evoke the question of how the toxin is distributed within a food sample, especially cow milk. Milk samples with different milk fat contents were incubated with purified cereulide, separated by centrifugation into a lipid and an aqueous phase, and cereulide was quantified in both fractions by SIDA-LC-MS/MS. By artificially increasing the milk fat content from 0.5% to 50%, the amount of cereulide recovered in the lipid phase and could be augmented from 13.3 to 78.6%. Further, the ratio of cereulide increased in the lipid phase of milk with additional plant-based lipid (sunflower oil) to 47.8%. This demonstrated a clear affinity of cereulide towards the hydrophobic, lipid phase, aligning with cereulide's naturally strong hydrophobic properties. Therefore, an intensified cereulide analysis of lipid enriched dairy products to prevent severe cereulide intoxications or cross-contamination in processed foods is suggested.

Multianalyte method for the determination of regulated, emerging and modified mycotoxins in milk: QuEChERS extraction followed by UHPLC-MS/MS analysis.

A simple method for the quantification of 40 mycotoxins in milk was developed. This method is based on a QuEChERS extraction followed by the ultra-high liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) detection, and allows the simultaneous analysis of regulated, emerging, and modified mycotoxins. A sample treatment procedure was optimized to include a concentration step for the analysis of some compounds such as aflatoxin M1. The method was in-house validated in terms of limits of detection (LODs), limits of quantification (LOQs), linearity, recoveries, and precision. LOQs lower than 10 ng/mL were obtained, and recoveries ranged from 61% to 120% with a precision, expressed as the relative standard deviation, lower than 15%. Therefore, acceptable performance characteristics were obtained fulfilling European regulations. The method was successfully applied for the quantification of mycotoxins in raw milk. It can be highlighted high occurrence of beauvericin and enniatins were found in low amounts.

Genomics assisted functional characterization of Paenibacillus polymyxa HK4 as a biocontrol and plant growth promoting bacterium.

The diseases caused by phytopathogens account for huge economic losses in the agricultural sector. Paenibacillus polymyxa is one of the agriculturally important biocontrol agents and plant growth promoting bacterium. This study describes the antifungal potential of P. polymyxa HK4 against an array of fungal phytopathogens and its ability to stimulate seed germination of cumin and groundnut under in vitro conditions. The cumin and groundnut seeds bacterized with HK4 exhibited enhanced germination efficiency in comparison to controls. The use of HK4 as a soil inoculant significantly promoted the shoot length and fresh weight of groundnut plants in pot studies. The draft genome analysis of HK4 revealed the genetic attributes for motility, root colonization, antagonism, phosphate solubilization, siderophore production and production of volatile organic compounds. The bacterium HK4 harnessed several hydrolytic enzymes that may assist its competence in the rhizosphere. The PCR amplification and sequence analysis of the conserved region of the fusA gene amplicon revealed the ability of HK4 to produce fusaricidin. Furthermore, the LC-ESI-MS/MS of crude cell pellet extract of HK4 confirmed the presence of fusaricidin as a major antifungal metabolite. This study demonstrated the potential of HK4 as a biocontrol agent and a plant growth promoter.

Acute Liver Failure after Ingestion of Fried Rice Balls: A Case Series of Bacillus cereus Food Poisonings.

Bacillus cereus foodborne intoxications and toxicoinfections are on a rise. Usually, symptoms are self-limiting but occasionally hospitalization is necessary. Severe intoxications with the emetic Bacillus cereus toxin cereulide, which is notably resistant heat and acid during cooking, can cause acute liver failure and encephalopathy. We here present a case series of food poisonings in five immunocompetent adults after ingestion of fried rice balls, which were massively contaminated with Bacillus cereus. The patients developed a broad clinical spectrum, ranging from emesis and diarrhoea to life-threatening acute liver failure and acute tubular necrosis of the kidney in the index patient. In the left-over rice ball, we detected 8 × 106Bacillus cereus colony-forming units/g foodstuff, and cereulide in a concentration of 37 µg/g foodstuff, which is one of the highest cereulide toxin contaminations reported so far from foodborne outbreaks. This report emphasizes the potential biological hazard of contaminated rice meals that are not freshly prepared. It exemplifies the necessity of a multidisciplinary approach in cases of Bacillus cereus associated food poisonings to rapidly establish the diagnosis, to closely monitor critically ill patients, and to provide supportive measures for acute liver failure and-whenever necessary-urgent liver transplantation.

Fusarium Cyclodepsipeptide Mycotoxins: Chemistry, Biosynthesis, and Occurrence.

Most of the fungi from the Fusarium genus are pathogenic to cereals, vegetables, and fruits and the products of their secondary metabolism mycotoxins may accumulate in foods and feeds. Non-ribosomal cyclodepsipeptides are one of the main mycotoxin groups and include beauvericins (BEAs), enniatins (ENNs), and beauvenniatins (BEAEs). When ingested, even small amounts of these metabolites significantly affect human and animal health. On the other hand, in view of their antimicrobial activities and cytotoxicity, they may be used as components in drug discovery and processing and are considered as suitable candidates for anti-cancer drugs. Therefore, it is crucial to expand the existing knowledge about cyclodepsipeptides and to search for new analogues of these compounds. The present manuscript aimed to highlight the extensive variability of cyclodepsipeptides by describing chemistry, biosynthesis, and occurrence of BEAs, ENNs, and BEAEs in foods and feeds. Moreover, the co-occurrence of Fusarium species was compared to the amounts of toxins in crops, vegetables, and fruits from different regions of the world.

Beauvericin and Enniatins: In Vitro Intestinal Effects.

Food and feed contamination by emerging mycotoxins beauvericin and enniatins is a worldwide health problem and a matter of great concern nowadays, and data on their toxicological behavior are still scarce. As ingestion is the major route of exposure to mycotoxins in food and feed, the gastrointestinal tract represents the first barrier encountered by these natural contaminants and the first structure that could be affected by their potential detrimental effects. In order to perform a complete and reliable toxicological evaluation, this fundamental site cannot be disregarded. Several in vitro intestinal models able to recreate the different traits of the intestinal environment have been applied to investigate the various aspects related to the intestinal toxicity of emerging mycotoxins. This review aims to depict an overall and comprehensive representation of the in vitro intestinal effects of beauvericin and enniatins in humans from a species-specific perspective. Moreover, information on the occurrence in food and feed and notions on the regulatory aspects will be provided.

Application of untargeted tandem mass spectrometry with molecular networking for detection of enniatins and beauvericins from complex samples.

An untargeted LC-MS/MS-based molecular networking method was established for the automatic determination of variants of enniatin and beauvericin from both fungal cultures and naturally contaminated samples. Using this method, a large number of samples can be efficiently analyzed for the presence of enniatin- and beauvericin-related compounds. As proof of concept, 26 cultures, derived from 13 fungal strains in the genera of Fusarium, Beauveria, and Diaporthe, as well as 46 food samples were analyzed. Four enniatin- and three beauvericin-producing fungi were newly discovered. Among them, the production of beauvericin by Fusarium sp. 190-20-2 was further confirmed by the presence of a beauvericin biosynthesis gene cluster in its genomic sequence. Additionally, 17 enniatin congeners, including one new isomer of enniatin A, and three previously unreported bassianolide analogues were detected from an enniatin-producing fungus, Fusarium sp. 17-048, and a beauvericin-producing fungus, Beauveria sp. 186-069, respectively. The structures of the detected compounds were tentatively determined by a series of product ions of their sodium adducts. The new isomer of enniatin A was further confirmed by NMR spectra. A preliminary survey of food samples showed that enniatins were prevalent in the tested wheat flour and noodle samples, whereas beauvericin was only discovered in cornflour powder samples.

Carry-over of some Fusarium mycotoxins in tissues and eggs of chickens fed experimentally mycotoxin-contaminated diets.

Fusarium mycotoxins are fungal contaminants found in different crops intended for human and animal consumption. Due to the co-occurrence of several of mycotoxins, the present study aimed at examining the transfer of these toxins into tissues of broiler chickens and eggs of laying hens fed contaminated diets. After an adaptation period, the chickens were fed contaminated diets containing mg/kg levels of deoxynivalenol (DON), enniatins (ENN A, A1, B, B1) and beauvericin (BEA) and high µg/kg levels of HT-2 toxin (HT-2), T-2 toxin (T-2) and zearalenone (ZEN) during a repletion period of two weeks, followed by a depletion period of two weeks. DON, ZEN, T-2 and HT-2 were not carried out into the skin and the liver of broiler chickens. ENN B (20.5 ± 6.6 µg/kg) and BEA (162 ± 55 µg/kg) were found in the liver, while in the skin their respective concentrations were 50 ± 17 µg/kg and 120 ± 16 µg/kg during the first week of the repletion period. Carry-over rates into liver and skin were higher for BEA (1.6% and 1.2%, respectively) than for ENNs (0.1 and 0.4%, respectively). During the depletion period, ENNs and BEA were eliminated from the skin and the liver. ENN B, ENN B1 and BEA were carried over into eggs at 0.1%, 0.05% and 0.44% upon 2-3 days of feeding the contaminated diet, respectively. These transfers were fully eliminated 9-10 days after feeding the control diet again. These results indicate the transfer of ENN B, ENN B1 and BEA from feed to chicken offal, meat products and eggs at a very low degree, thus marginally contribute to the total dietary intake of these fusariotoxins for consumers. Nevertheless, taking precautionary measures in the field, harvest, transport and storage of the raw materials is required to keep the mycotoxin concentration in feed below the safe levels.

Fumonisin and Beauvericin Chemotypes and Genotypes of the Sister Species Fusarium subglutinans and Fusarium temperatum.

Fusarium subglutinans and Fusarium temperatum are common maize pathogens that produce mycotoxins and cause plant disease. The ability of these species to produce beauvericin and fumonisin mycotoxins is not settled, as reports of toxin production are not concordant. Our objective was to clarify this situation by determining both the chemotypes and genotypes for strains from both species. We analyzed 25 strains from Argentina, 13 F. subglutinans and 12 F. temperatum strains, for toxin production by ultraperformance liquid chromatography mass spectrometry (UPLC-MS). We used new genome sequences from two strains of F. subglutinans and one strain of F. temperatum, plus genomes of other Fusarium species, to determine the presence of functional gene clusters for the synthesis of these toxins. None of the strains examined from either species produced fumonisins. These strains also lack Fum biosynthetic genes but retain homologs of some genes that flank the Fum cluster in Fusarium verticillioides None of the F. subglutinans strains we examined produced beauvericin although 9 of 12 F. temperatum strains did. A complete beauvericin (Bea) gene cluster was present in all three new genome sequences. The Bea1 gene was presumably functional in F. temperatum but was not functional in F. subglutinans due to a large insertion and multiple mutations that resulted in premature stop codons. The accumulation of only a few mutations expected to disrupt Bea1 suggests that the process of its inactivation is relatively recent. Thus, none of the strains of F. subglutinans or F. temperatum we examined produce fumonisins, and the strains of F. subglutinans examined also cannot produce beauvericin. Variation in the ability of strains of F. temperatum to produce beauvericin requires further study and could reflect the recent shared ancestry of these two species.IMPORTANCEFusarium subglutinans and F. temperatum are sister species and maize pathogens commonly isolated worldwide that can produce several mycotoxins and cause seedling disease, stalk rot, and ear rot. The ability of these species to produce beauvericin and fumonisin mycotoxins is not settled, as reports of toxin production are not concordant at the species level. Our results are consistent with previous reports that strains of F. subglutinans produce neither fumonisins nor beauvericin. The status of toxin production by F. temperatum needs further work. Our strains of F. temperatum did not produce fumonisins, while some strains produced beauvericin and others did not. These results enable more accurate risk assessments of potential mycotoxin contamination if strains of these species are present. The nature of the genetic inactivation of BEA1 is consistent with its relatively recent occurrence and the close phylogenetic relationship of the two sister species.

Mycotoxins in maize harvested in Serbia in the period 2012-2015. Part 2: Non-regulated mycotoxins and other fungal metabolites.

The main objective of this study was to screen, for the first time, the natural occurrence of non-regulated fungal metabolites in 204 maize samples harvested in Serbia in maize growing seasons with extreme drought (2012), extreme precipitation and flood (2014) and moderate drought conditions (2013 and 2015). In total, 109 non-regulated fungal metabolites were detected in examined samples, whereby each sample was contaminated between 13 and 55 non-regulated fungal metabolites. Moniliformin and beauvericin occurred in all samples collected from each year. In samples from year 2012, oxaline, questiomycin A, cyclo (l-Pro-l-Val), cyclo (l-Pro-l-Tyr), bikaverin, kojic acid and 3-nitropropionic acid were the most predominant (98.0-100%). All samples from 2014 were contaminated with 7-hydroxypestalotin, 15-hydroxyculmorin, culmorin, butenolid and aurofusarin. Bikaverin and oxaline were quantified in 100% samples from 2013 and 2015, while 3-nitropropionic acid additionally occurred in 100% samples from 2015.

A multi-year survey of mycotoxins and ergosterol in Canadian oats.

Canadian oat harvest samples, deliveries to processors, and train shipments from primary elevators were collected from mid-2014 through mid-2017 and analyzed for 26 mycotoxins and the fungal biomarker ergosterol. Of the 26 mycotoxins, 7 were not detected in any sample. The most frequently measured mycotoxins were beauvericin (in over 95% of samples analyzed), followed by tentoxin, culmorin, alternariol, alternariol methyl ether, and deoxynivalenol. Median concentrations of the Fusarium-produced mycotoxins ranged from 68 to 1142 µg/kg for deoxynivalenol, 39 to 188 µg/kg for HT-2 and T-2 toxins, 66 to 232 µg/kg for nivalenol, and less than 35 µg/kg for beauvericin. Median concentrations of the sum of Alternaria-produced mycotoxins were all less than 250 µg/kg. Concentrations of analytes varied among years, as well as among growing areas, for the harvest samples. Ergosterol, Fusarium, and Alternaria mycotoxin concentrations appeared to increase from the west toward the eastern Prairies and the province of Quebec; the differences were not statistically significant though. Ochratoxin A in deliveries and train shipments showed annual cyclic increases in the late summer. The results of the survey demonstrate the general compliance of Canadian oats with existing maximum levels for mycotoxins and indicate that in late summer and in years with increased Fusarium infection, there can be a need for monitoring of ochratoxin A and deoxynivalenol, respectively, to mitigate risks of noncompliant grain.

Plant-based milks: unexplored source of emerging mycotoxins. A proposal for the control of enniatins and beauvericin using UHPLC-MS/MS.

Mycotoxins have become one of the most common contaminants reported worldwide. Current legislation has established maximum levels only for some well-known mycotoxins; however, there are many other "emerging mycotoxins" for which there is no regulation, as enniatins and beauvericin. An analytical method based on salting-out assisted liquid-liquid extraction followed by ultra-high performance liquid chromatography tandem mass spectrometry is proposed for determination of enniatin A, A1, B, B1, and beauvericin in different plant-based milks, as a possible source of these contaminants, is proposed. The method showed good precision and trueness (RSD <8% and recoveries between 84-97%) with a moderate matrix effect. From a total of 32 samples of plant-based milks of different compositions (including 8 rice milks, 8 oat milks and 16 soy milks), 3 samples were contaminated with the five mycotoxins, while 5 samples were contaminated with four of them, being oat milk the most susceptible for contamination.

Fusarium species and enniatin mycotoxins in wheat, durum wheat, triticale and barley harvested in France.

Contamination with enniatins A, A1, B and B1 was investigated in 1240 samples of small grain cereals (470 wheat, 260 durum wheat, 282 spring barley, 172 triticale and 56 winter barley) from the French harvests of 2012 to 2014. Associations with Fusarium avenaceum, F. tricinctum and F. poae were assessed, with the identification of Fusarium species by real-time PCR and mycotoxin quantification by LC-MS/MS. Enniatins were common in the fields sampled. Enniatin concentrations varied between years but were consistently highest on spring barley (mean values of 199 to 1316 µg/kg) and triticale (mean values from 131 to 1218 µg/kg), and lower on wheat (mean values from 47 to 142 µg/kg) and durum wheat (mean values from 55 to 596 µg/kg). The concentrations of the various enniatins were strongly correlated with each other (Pearson's correlation coefficient of 0.61 to 0.98). Enniatin B was the most frequent (68% of the total enniatin content), followed by enniatin B1 (22%), enniatin A1 (7%) and enniatin A (3%). Fusarium species were quantified by calculating arithmetic mean total DNA levels. F. tricinctum was the most abundant (0.177 pg/ng total DNA), followed by F. avenaceum (0.141 pg/ng total DNA) and F. poae (0.091 pg/ng total DNA). Total DNA levels for each species, and the predominant species varied between years and crops. Small grain cereal species (p value < 0.001), harvest year (p value = < 0.001) and the presence of F. avenaceum (p value < 0.001), F. tricinctum (p value < 0.001) or F. poae (p value = 0.017) affected enniatin content. F. tricinctum was the leading enniatin producer on durum wheat (29% to 45%) and spring barley (23 to 37%). F. avenaceum produced large amounts of enniatins on durum wheat (13% to 17%) and wheat (1% to 18%) and was the leading producer on triticale (30% to 55%). F. poae made a minor contribution on all crops, never accounting for more than 2% of total enniatin content. Enniatins are, thus, highly prevalent in French small grain cereals and are mostly produced by F. avenaceum and F. tricinctum.

Rapid detection of Bacillus ionophore cereulide in food products.

Cereulide is a toxic cyclic depsipeptide produced by certain strains of Bacillus cereus found in soil and food products. While some harmless strains of Bacillus are used as probiotic, others can cause nausea and vomiting, and represent an important food safety concern. Current detection methods are time consuming and do not necessarily detect toxic cereulide. Here, we developed a rapid protocol using Matrix Assisted Laser Desorption/Ionization-Time of Flight (MALDI-TOF) mass spectrometry that detects the toxin originating from a colony smear of B. cereus. The distinct molecular feature of the toxin peak at m/z 1,191 was clearly identified from bacterial extracts with a limit of detection (LOD) of 30 ng/mL. Final optimisation of the sample preparation was based on cereulide chelating cations to produce the alkali adduct [M + K]+ without the use of a MALDI matrix, and provided a 1,000-fold improvement of LOD with 30 pg/mL of cereulide. We evaluated the application of this method for the detection of cereulide in rice, milk, and different ready-to-eat meals. The proposed protocol is quick, easy and provides an improvement over conventional methods for the detection of B. cereus toxin.

Identification of autoinducing thiodepsipeptides from staphylococci enabled by native chemical ligation.

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.

Occurrence of Mycotoxigenic Fusarium Species and Competitive Fungi on Preharvest Maize Ear Rot in Poland.

Maize has become one of the most important crops for food and feed production-both as a silage and crop residue worldwide. The present study aimed to identify the co-occurrence of Fusarium subglutinans, Fusarium verticillioides, Trichoderma atroviride, Sarocladium zeae, and Lecanicillium lecanii on maize ear rot. Further, the accumulation of mycotoxins as secondary metabolites of Fusarium spp. in maize ear samples was also analyzed. Maize ear samples were collected between 2014 and 2017 from two main maize growing areas in Poland (Greater Poland and Silesia region). A significant difference was found in the frequency of two main Fusarium spp. that infect maize ears, namely F. subglutinans and F. verticillioides. In addition to Fusarium spp. T. atroviride, S. zeae, and L. lecanii were also identified. T. atroviride species was found in 14% of maize samples examined between 2014 and 2017, particularly with a high percentage of Trichoderma spp. recorded in 2014, i.e., in 31% of samples. However, mycotoxin content (beauvericin and fumonisins) varied, depending on both the location and year of sampling. The interaction of fungi and insects inhabiting maize ear and kernel is very complex and not yet elucidated. Therefore, further research is required in this area.