Food poisoning toxins fingered
[December 1, 2008]

Source of Article:

One of the most common food-borne diseases is staphylococcal food poisoning, caused by the bacterium Staphylococcus aureus. It accounts for hundreds of thousands of cases each year and vies with Salmonella for top spot in the food poisoning league table. The individual organisms exist in pairs and in grape-like clusters, which gave rise to the name: the Greek word staphyle means a bunch of grapes.

Attractive though the nomenclature is, the bug is less alluring, provoking serious vomiting and diarrhoea for up to eight hours after ingestion. It has also spawned a form known as methicillin-resistant S. aureus (MRSA) which has played havoc in hospitals, resulting in the deaths of vulnerable patients.

The active staphylococcal agents are a set of proteins called enterotoxins, 19 of which have been identified to date. However, the shortage of specific diagnostic tools for many of these toxins means that a significant number of suspected staphylococcal food poisonings have not been proven. The classical methods involve ELISA immunological tests for just five of the staphylococcal agents (A, B, C, D and E). Even then, the tests have serious drawbacks, which have been summarised by a team of French researchers.

Virginie Brun, Alain Dupuis and Jerome Garin, all affiliated to the three Grenoble organisations of the Institute of Life Sciences Research and Technologies, INSERM U880 and the University of Joseph Fourier, and Jacques-Antoine Hennekinne from the French Agency for Food Safety, recognised three problems. Very few specific antibodies are available due to the close structural similarity between enterotoxins. This is compounded by the occurrence of some non-specific reactions from food matrices and interference from a common immunoglobulin-binding protein that is found in foods.

The French solution approached the enterotoxin identification problem from a different angle, with mass spectrometry. They used a technique which they announced in 2007 called Protein Standard Absolute Quantification (PSAQ) which determines the absolute amounts of protein by the isotope dilution technique. However, rather than adding isotopically labelled peptides as other protein quantifications have done, they added isotopically labelled versions of the complete enterotoxins which they synthesised on site. This has the marked advantage that the endogenous toxins and the structurally identical standards (apart from the labels) have identical biochemical properties and will react the same way throughout their treatment.

Their method was illustrated with a semi-hard cheese prepared from cow's milk that had been inoculated with a strain of S. aureus producing staphylococcal enterotoxin A (SEA). A portion of the cheese was depleted of the abundant caseins and dialysed before the addition of an isotopically labelled SEA. The extract was enriched on an immunoaffinity column designed to capture SE(A to E) and the eluates subjected to SDS-PAGE. The SE regions of the gel were cut out and digested with trypsin for analysis by electrospray ionisation LC/MS.

The selectivity of mass spectrometry permits the identification of peptides that are specific to one particular endotoxin. In this case, a decapeptide characteristic of SEA was found in the cheese sample, pinpointing the presence of endogenous SEA. The amount of the endotoxin was determined by comparing the integrated peak of the endogenous and labelled peptides giving a concentration of 2.5 ng/g. This value was consistent with that obtained by ELISA at 2.9 ng/g.

Following this proof of concept, the method was applied to a naturally contaminated Chinese dessert that was the focus of a food poisoning outbreak in France in 2006 in which a S. aureus strain encoding SEA was detected. The sample was spiked with the SEA labelled standard and processed as before. In this case, two endogenous peptides from SEA were found, the decapeptide found in the cheese sample as well as a second peptide. They were used to determine SEA at 1.47 ng/g, the values obtained from each peptide differing by 0.7%.

Brun demonstrated the potential of the technique by analysing the tryptic digests of 13 staphylococcal enterotoxins by mass spectrometry. The resulting panel of peptides revealed specific marker compounds for all 13 toxins.

The sensitivity of the overall method could be increased by using multiple reaction monitoring and this might reveal some of the peptides from the 13 enterotoxins that were predicted but not observed, possibly due to their low levels. Although the performance promises to be more accurate than ELISA, the unit cost and throughput time of an analysis are comparatively unfavourable. However, the researchers argued that it takes up to a year and around one million dollars to develop an ELISA method. In addition, one batch of labelled protein would suffice for thousands of quantitative analyses.

The team are now synthesising a library of isotopically labelled staphylococcal enterotoxins and hope to instigate a "community-based effort to develop large PSAQ libraries for protein biomarkers." In the meantime, they believe that the detection of staphylococcal food poisoning incidents will be facilitated by the PSAQ method, identifying cases where ELISA will fail.

Related links:

  • Proteomics 2008, 8, 4633-4666: "Protein Standard Absolute Quantification (PSAQ) for improved investigation of staphylococcal food poisoning outbreaks"

Article by Steve Down


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