RESUMEN
In the frame of the CEN Mandate M/381 from the European Commission to CEN (European Committee for Standardization), a method for the detection of staphylococcal enterotoxins in foodstuffs has been developed, validated and standardized. An extraction procedure based on dialysis concentration followed by an immuno-enzymatic detection has been defined. In addition, performance criteria (minimum values of sensitivity, specificity and level of detection) to be achieved by the commercially available immuno-enzymatic kits that could be used to detect staphylococcal enterotoxins in food matrices, were developed. A 2-stage validation study was conducted: The first stage aimed at selecting the commercial kits to be included in the second stage, which consisted in an interlaboratory study, using eight matrices covering five food categories (ready-to-eat food, meat products, milk products, dessert and fish). Results showed that two detection kits included in the study met the pre-defined performance criteria. The implementation of dialysis concentration step increased significantly the sensitivity of the method. The method developed allowed to achieve the Benchmark Dose lower limit (BMD10) estimated at 6.1 ng. In 2019, finally, the European Commission recognized this standard as the European Union reference method for the detection of staphylococcal enterotoxins in food.
Asunto(s)
Enterotoxinas , Análisis de los Alimentos , Microbiología de Alimentos , Animales , Enterotoxinas/análisis , Unión Europea , Análisis de los Alimentos/métodos , Cadena Alimentaria , Microbiología de Alimentos/métodos , Límite de DetecciónRESUMEN
Currently, only 5 (SEA to SEE) out of 27 known staphylococcal enterotoxins can be analyzed using commercially available kits. Six genes (seg, sei, sem, sen, seo, and seu), encoding putative and undetectable enterotoxins, are located on the enterotoxin gene cluster (egc), which is part of the Staphylococcus aureus genomic island vSaß. These enterotoxins have been described as likely being involved in staphylococcal food-poisoning outbreaks. The aim of the present study was to determine if whole-genome data can be used for the prediction of staphylococcal egc enterotoxin production, particularly enterotoxin G (SEG) and enterotoxin I (SEI). For this purpose, whole-genome sequences of 75 Staphylococcus aureus strains from different origins (food-poisoning outbreaks, human, and animal) were investigated by applying bioinformatics methods (phylogenetic analysis using the core genome and different alignments). SEG and SEI expression was tested in vitro using a sandwich enzyme-linked immunosorbent assay method. Strains could be allocated to 14 different vSaß types, each type being associated with a single clonal complex (CC). In addition, the vSaß type and CC were associated with the origin of the strain (human or cattle derived). The amount of SEG and SEI produced also correlated with the vSaß type and the CC of a strain. The present results show promising indications that the in vitro production of SEG and SEI can be predicted based on the vSaß type or CC of a strain. IMPORTANCE Besides having infectious properties in human and animals, S. aureus can produce different enterotoxins in food. The enterotoxins can cause vomiting and diarrhea, often involving many people. Most of these outbreaks remain undiscovered, as detection methods for enterotoxins are only available for a few enterotoxins but not for the more recently discovered enterotoxins G (SEG) and I (SEI). In this study, we show promising results that in vitro production of SEG and SEI can be predicted based on the whole-genome sequencing data of a strain. In addition, these data could be used to find the source (human or cattle derived) of an outbreak strain, which is the key for a better understanding of the role SEG and SEI play in foodborne outbreaks caused by S. aureus.
Asunto(s)
Enterotoxinas , Enfermedades Transmitidas por los Alimentos , Staphylococcus aureus , Animales , Técnicas de Tipificación Bacteriana , Bovinos , Enterotoxinas/genética , Enfermedades Transmitidas por los Alimentos/epidemiología , Humanos , Familia de Multigenes , Filogenia , Staphylococcus aureus/clasificación , Staphylococcus aureus/genéticaRESUMEN
AIM: To determine the performance of the Ridascreen® SET Total kit, after sample extraction and concentration by dialysis, with regard to its use in official controls for staphylococcal enterotoxins under European Regulation (EC) No. 2073/2005 modified. This study was conducted on naturally contaminated cheese samples and compared with the results of the previously validated Vidas® SET2 kit. METHODS AND RESULTS: The effectiveness of the Ridascreen® SET Total kit on naturally contaminated cheeses was compared to that of the Vidas® SET2 kit by applying the EN ISO 16140 standard. Sensitivity and specificity were also compared using spiked buffer solutions and cheese samples with SEA to SEE toxins. CONCLUSIONS: This study showed that the Ridascreen® SET Total kit is as effective as the Vidas® SET2 kit. SIGNIFICANCE AND IMPACT OF THE STUDY: The Ridascreen® SET Total kit was found to specifically detect SEA to SEE in cheeses. The Ridascreen® SET Total can therefore be used to check the staphylococcal enterotoxin content and ensure consumer protection.
Asunto(s)
Queso/análisis , Enterotoxinas/análisis , Microbiología de Alimentos/métodos , Juego de Reactivos para Diagnóstico/normas , Sensibilidad y Especificidad , Staphylococcus/químicaRESUMEN
Two different immunoaffinity columns (IACs) were prepared for detection of staphylococcal enterotoxins (SETs) from dairy products. First, a specific IAC for staphylococcal enterotoxin A (SEA), IAC-1, was prepared by coupling monoclonal antibody (mAb) directed against SEA; second, a polyspecific IAC for SEA, staphylococcal enterotoxin B (SEB), staphylococcal enterotoxin C (SECs), and staphylococcal enterotoxin D (SED), IAC-2, was prepared by coupling a mixture of mAbs against SEA, SECs, and SED, and rabbit IgG against SEB. These columns were applied for detection of SETs in dairy products, after extraction, immunoaffinity chromatography, and enzyme immunosorbent assay (EIA). Overall recoveries from dairy products spiked with 1 ng SEA/25 g averaged 81.2% (range, 76-85%) on IAC-1. The repeated use of IAC-1 was then determined with good efficiency of 91.5%, in more than 10 runs. On the other hand, a recovery yield of 77% of SETs (SEA, SEB, SEC, and SED) from dairy products spiked with 2.5 ng of each enterotoxin per 25 g, was obtained with IAC-2. IAC-2 was also successfully subjected to the chromatography of naturally contaminated foods implicated in staphylococcal food poisoning outbreaks. This new extraction-concentration-immunoaffinity-chromatography method (ECIC) is very useful for improving staphylococcal enterotoxin detection and eliminating matrix effect in EIA of dairy products.