Quantitative Determination of Staphylococcus aureus Enterotoxins Types A to I and Variants in Dairy Food Products by Multiplex Immuno-LC-MS/MS

Abstract

Staphylococcal enterotoxins (SEs) are responsible for frequent food poisoning outbreaks worldwide. Specific identification of SEs is crucial for confirmation of food poisoning, tracking of the incriminated foods or food ingredients, and removal from the food chain. Here, we report on a new food testing protocol addressing the challenge of low abundance of SEs in contaminated food and high sequence heterogeneity. Multiplex ability of targeted high-resolution mass spectrometry was succesfully applied to the simultaneous and quantitative determination of the eight most frequent SEs including sequence variants. In this aim, between three and eight proteotypic peptides of each SE were selected by carefully considering amino acid variations within each type, and sequence homology between types. Quantification of trace levels of SEs directly in food samples was reached by immunoaffinity enrichment and optimized analytical conditions. The assay was validated in dairy food products with a lower limit of quantification down to 0.1 ng/g (in milk), and quantification of SEs was successfully demonstrated in real-life samples collected during staphylococcal food poisoning outbreaks. Importantly, the ability of the method to detect diverse sequence variants was also illustrated. By enabling for the first time the simultaneous quantification of the eight most frequent SEs, the new mass spectrometry-based assay would facilitate the laboratory confirmation of positive samples in situation of food poisoning outbreaks.

Development of a Double Nanobody-Based Sandwich Immunoassay for the Detecting Staphylococcal Enterotoxin C in Dairy Products

“…Therefore, SEs remain active and pathogenic after treatment with general methods, with only a few micrograms necessary to cause SFP. In Europe, about 15-20% of food poisoning outbreaks (FPO) are caused by bacterial toxins, of which about 50% are related to the SEs produced by S. aureus [2]. The main SE serotypes include SEA, SEB, SECs, SED, and SEE.…”

Top-Down Mass Spectrometry for Trace Level Quantification of Staphylococcal Enterotoxin A Variants

“…Enrichment of SEA prior to mass spectrometry using specific antibodies was considered as the most appropriate procedure to reach low toxin amounts in bacteria culture medium or food samples. Conditions were similar to those of our previous work 8 using immunoaffinity enrichment by two specific antibodies coated on magnetic beads. A mixture of monoclonal antibodies with different epitopes was shown to be beneficial for efficient capture of sequence variants 8 and this was verified here by the detection of the four SEA variants in the culture supernatant (Figure S3, Table S3).…”

“…Conditions were similar to those of our previous work 8 using immunoaffinity enrichment by two specific antibodies coated on magnetic beads. A mixture of monoclonal antibodies with different epitopes was shown to be beneficial for efficient capture of sequence variants 8 and this was verified here by the detection of the four SEA variants in the culture supernatant (Figure S3, Table S3). After capture and washing, SEA was eluted from the beads by an optimized mixture of water/acetonitrile (80/20) containing 1% HCOOH and 0.002% bovine serum albumin (Figure S4).…”

“…7 SEA toxin standard was obtained from Toxin Technology (Sarasota, FL) and previously characterized. 8 Brain heart infusion (BHI) medium was from Oxoid Thermo Fisher Scientific (Waltham, MA). BHI was processed on 0.2 μm filters 16534K from Sartorius (Goẗtingen, Germany).…”

“…12−14 In this way, we recently reported the detection and quantification of several types of SEs by monitoring canonic peptides representative of the toxin. 8 Although the bottom-up assay was able to detect all reported sequence variants, identification and discrimination of the variant involved in the FPO was not achieved. Bottom-up assays are indeed not well suited for discrimination between sequence variants due to only partial sequence coverage and loss of information on sequence modifications.…”