Detection of toxins involved in foodborne diseases caused by Gram-positive bacteria.

Bacterial toxins are food safety hazards causing about 10% of all reported foodborne outbreaks in Europe. Pertinent to Gram-positive pathogens, the most relevant toxins are emetic toxin and diarrheal enterotoxins of Bacillus cereus, neurotoxins of Clostridium botulinum, enterotoxin of Clostridium perfringens, and a family of enterotoxins produced by Staphylococcus aureus and some other staphylococci. These toxins are the most important virulence factors of respective foodborne pathogens and a primary cause of the related foodborne diseases. They are proteins or peptides that differ from each other in their size, structure, toxicity, toxicological end points, solubility, and stability, types of food matrix to which they are mostly related to. These differences influence the characteristics of required detection methods. Therefore, detection of these toxins in food samples, or detection of toxin production capacity in the bacterial isolate, remains one of the cornerstones of microbial food analysis and an essential tool in understanding the relevant properties of these toxins. Advanced research has led into new insights of the incidence of toxins, mechanisms of their production, their physicochemical properties, and their toxicological mode of action and dose-response profile. This review focuses on biological, immunological, mass spectrometry, and molecular assays as the most commonly used detection and quantification methods for toxins of B. cereus, C. botulinum, C. perfringens, and S. aureus. Gathered and analyzed information provides a comprehensive blueprint of the existing knowledge on the principles of these assays, their application in food safety, limits of detection and quantification, matrices in which they are applicable, and type of information they provide to the user.

Counteracting in Vitro Toxicity of the Ionophoric Mycotoxin Beauvericin-Synthetic Receptors to the Rescue.

Beauvericin (BEA) and enniatins are toxic ionophoric cyclodepsipeptides that mainly occur in grains. As such, their presence in food commodities poses a concern for public health. To date, despite recent European Food Safety Authority emphasis on the need for more data to evaluate long-term toxicity effects, no suitable affinity reagents are available to detect the presence of BEA and derivatives in food samples. We here report on the synthesis of a small library of artificial receptors with varying cavity sizes and different hydrophobic building blocks. Immobilization of one of the receptors on solid support resulted in a strong retention of beauvericin, thus revealing promising properties as solid-phase extraction material for sample pretreatment. Furthermore, treatment of HepG2 cells with the most promising receptor markedly reduced beauvericin-induced cytotoxicity, hinting toward the possibility of using synthetic receptors as antidotes against ionophoric toxins.

Impact of fungicides and weather on cyclodepsipeptide-producing Fusarium spp. and beauvericin and enniatin levels in wheat grains.

BACKGROUND: Fusarium head blight (FHB) is a well-known disease of wheat caused by a complex of Fusarium species. In this research, an extensive study on the occurrence of the emerging Fusarium cyclodepsipeptide mycotoxins beauvericin and enniatins was conducted in Belgian wheat grains harvested in 2015 and 2016. To assess the link between Fusarium species and their mycotoxin production, ultra-performance liquid chromatography-tandem mass spectrometry was used to quantify the cyclodepsipeptide mycotoxins, while quantitative polymerase chain reaction was applied to quantify the presence of Fusarium species. RESULTS: It was shown that enniatins were mainly associated with the presence of F. avenaceum, while beauvericin, despite its low incidence, correlated significantly with F. poae. The application of fungicides resulted in a species shift and in the occurring mycotoxins. Concerning the effect of weather conditions, it was seen that levels of enniatins were positively correlated with the rainfall in May and June, while a negative correlation was observed with rainfall in the first half of July. CONCLUSION: Our study provides new insights into the occurrence of the emerging cyclodepsipeptide mycotoxins in an agro-ecosystem in which fungicides are the main control measure against FHB. It seems that beauvericin and enniatin levels are affected by different parameters and behave differently upon application of fungicides. © 2018 Society of Chemical Industry.

Oxygen Consumption Rate Analysis of Mitochondrial Dysfunction Caused by Bacillus cereus Cereulide in Caco-2 and HepG2 Cells.

The emetic syndrome of Bacillus cereus is a food intoxication caused by cereulide (CER) and manifested by emesis, nausea and in most severe cases with liver failure. While acute effects have been studied in the aftermath of food intoxication, an exposure to low doses of cereulide might cause unnoticed damages to the intestines and liver. The toxicity which relies on the mitochondrial dysfunction was assessed on Caco-2 and HepG2 cells after exposure of one, three and ten days to a range of low doses of cereulide. Oxygen consumption rate analyses were used to study the impact of low doses of CER on the bioenergetics functions of undifferentiated Caco-2 and HepG2 cells using Seahorse XF extracellular flux analyzer. Both Caco-2 and HepG2 cells experienced measurable mitochondrial impairment after prolonged exposure of 10 days to 0.25 nM of cereulide. Observed mitochondrial dysfunction was greatly reflected in reduction of maximal cell respiration. At 0.50 nM CER, mitochondrial respiration was almost completely shut down, especially in HepG2 cells. These results corresponded with a severe reduction in the amount of cells and an altered morphology, observed by microscopic examination of the cells. Accurate and robust quantification of basal respiration, ATP production, proton leak, maximal respiration, spare respiratory capacity, and non-mitochondrial respiration allowed better understanding of the effects of cereulide in underlying respiratory malfunctions in low-dose exposure.

Development and validation of ultra-high-performance liquid chromatography-tandem mass spectrometry methods for the simultaneous determination of beauvericin, enniatins (A, A1, B, B1) and cereulide in maize, wheat, pasta and rice.

Rapid and accurate UPLC-MS/MS methods for the simultaneous determination of beauvericin and the related enniatins (A, A1, B, B1), together with cereulide were successfully developed and validated in cereal and cereal-based food matrices such as wheat, maize, rice and pasta. Although these emerging foodborne toxins are of different microbial origin, the similar structural, toxicological and food safety features provided rationale for their concurrent detection in relevant food matrices. A Waters Acquity UPLC system coupled to a Waters Quattro Premier XE™ Mass Spectrometer operating in ESI+ mode was employed. Sample pretreatment involved a fast and simple liquid extraction of the target toxins without any further clean-up step. For all toxins the sample preparation resulted in acceptable extraction recoveries with values of 85-105% for wheat, 87-106% for maize, 84-106% for rice and 85-105% for pasta. The efficient extraction protocol, together with a fast chromatographic separation of 7min allowed substantial saving costs and time showing its robustness and performance. The validation of the developed method was performed based on Commission Decision 2002/657/EC. The obtained limits of detection ranged from 0.1 to 1.0µgkg-1 and the limits of quantification from 0.3 to 2.9µgkg-1 for the targeted toxins in the selected matrices. The obtained sensitivities allow detection of relevant toxicological concentrations. All relative standard deviations for repeatability (intra-day) and intermediate precision (inter-day) were lower than 20%. Trueness, expressed as the apparent recovery varied from 80 to 107%. The highly sensitive and repeatable validated method was applied to 57 naturally contaminated samples allowing detection of sub-clinical doses of the toxins.