Letters in Applied Microbiology ISSN 0266-8254

ORIGINAL ARTICLE

Intra-laboratory validation of the Ridascreen SET Total

kit for detecting staphylococcal enterotoxins SEA to
SEE in cheese
A. Ostyn, F. Guillier, A.-L. Prufer, I. Papinaud, S. Messio, S. Krys, B. Lombard and J.-A. Hennekinne
French Agency for Food, Environmental and Occupational Health & Safety (ANSES), Maisons-Alfort Laboratory for Food Safety,
Unit Characterization of Toxins, National and European Union Reference Laboratory for Coagulase Positive Staphylococci Including
Staphylococcus aureus, Maisons-Alfort Cedex, France

Keywords
cheese, EIA-based method, enterotoxins,
Staphylococcus aureus.
Correspondence
Jacques-Antoine Hennekinne, French Agency
for Food, Environmental and Occupational
Health & Safety (ANSES), Maisons-Alfort
Laboratory for Food Safety, 23 avenue du
Général de Gaulle, 94704 Maisons-Alfort
cedex, France.
E-mail: jacques-antoine.hennekinne@anses.fr
2010 ⁄ 2188: received 3 December 2010,
revised 3 December 2010 and accepted 28
January 2011
doi:10.1111/j.1472-765X.2011.03025.x
Abstract
Aim: To determine the performance of the Ridascreen SET Total kit, after
sample extraction and concentration by dialysis, with regard to its use in offi-
cial 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 effec-
tive 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.

food matrices involved in staphylococcal food poisoning

Introduction
Staphylococcal food poisoning is a human illness resulting
from the ingestion of staphylococcal enterotoxins (SEs)
preformed in foods by enterotoxigenic strains of coagu-
lase-positive staphylococci (CPS), such as Staphylococcus
aureus (Chokesajjawatee et al. 2009). Dairy products may
be involved in food-poisoning outbreaks because CPS is
known to occur in raw milk (Duquenne et al. 2010). In
2008, the European Food Safety Authority (Anon. 2010a)
reported that bacterial toxins were involved in 525 out of
5332 notified food-poisoning outbreaks (9Æ8%), ranking
third after Salmonella spp. (35Æ4%) and viruses (13Æ1%).
Among bacterial toxins, SEs have been implicated in 291
of the 525 notified food-poisoning outbreaks (or 55Æ4%),
thus representing 5Æ5% of all notified outbreaks. Among
outbreaks (SFPO), 7% were cheeses and 4% other dairy
products.
SE production generally occurs when the number of
staphylococci is higher than 105 colony-forming units
(CFU) per gram of cheese during the manufacturing pro-
cess. Various CPS biotypes can be found in milk, but ani-
mal biotypes are more frequently encountered than
human biotypes (Fedio et al. 2008).
To date, 21 staphylococcal enterotoxins (SEs) and
enterotoxin-like (SEl) types have been described: the first
five types to be described (SEA, SEB, SEC including vari-
ants SEC1, SEC2, SEC3 and ovine and bovine SEC, SED
and SEE) and 16 new SE types (SEG-SEI, SER-SET) and
SEl types (SElJ-SElQ, SElU and SElV) (Argudin et al.
2010; Hennekinne et al. 2010).

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468 Letters in Applied Microbiology 52, 468–474 ª 2011 The Society for Applied Microbiology
A. Ostyn et al.
The relationship between the newly described SEs ⁄ SEls
and food-poisoning outbreaks is still not very well fully
understood. Nevertheless these toxins may also be the
cause of illness (Ikeda et al. 2005; Jorgensen et al. 2005).
As low amounts of SEs are present in food vehicles
(Hennekinne et al. 2009; Ostyn et al. 2010) and given the
fact that many food samples contain various levels of pro-
teins, an extraction step must be performed before detect-
ing SEs. After this extraction step, enzyme immunoassay
(EIA) based-methods are generally used to detect SEs
(SEA to SED or SEA to SEE). Commercially available kits
are accepted as rapid and simple methods for detecting
these SEs. These kits have been developed according to
three different principles: ELISA, enzyme-linked fluores-
cent assay (ELFA) and reverse passive latex agglutination
(RPLA). To date, these kits have not been developed to
detect SEs other than the first five types, SEA to SEE.
However, due to their close primary sequences, the newer
SE types may cross-react with antibodies developed
against SEA to SEE.
The aim of this study was to determine the parameters
of performance of a new commercially available immuno-
logical kit, the Ridascreen SET Total (R-Biopharm,
Darmstadt, Germany), in milk and milk products, and
compare them to those of the Vidas SET2 kit (bio-
Mérieux, Marcy l’Etoile, France) in naturally contami-
nated cheese samples, using the protocol laid out in the
EN ISO 16140 Standard (Anon. 2003).
This study was conducted under our mandate as the
European Union-Reference Laboratory (EU-RL) and
National Reference Laboratory (NRL) for CPS for two
purposes: (i) to assess whether this kit can be used as a
rapid, specific and sensitive method for official controls
to guarantee a high level of public health protection and
(ii) to propose an alternative to the Vidas SET2 kit that
requires an automated instrument and has already been
validated for official controls.
Materials and Methods
Milk product samples
A total of 65 cheese samples were tested in this intra-lab-
oratory validation study. These cheeses were made from
cow’s (n = 27), goat’s (n = 11) and sheep’s milk (n = 6)
or with milk of unspecified origin (n = 21). All the natu-
rally contaminated cheese samples came from a collection
of food samples received since 2008 at the EU-RL for
CPS as part of official controls, internal checks, SFPO
characterization or research projects. All of them had
undergone a CPS count with results higher than
103 CFU g)1. A protein extraction step with subsequent
dialysis concentration, as recommended in the European
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Letters in Applied Microbiology 52, 468–474 ª 2011 The Society for Applied Microbiology
SEA to SEE detection in cheeses
screening method (ESM) (Anon. 2010b), was performed
before applying the immunological tests Vidas SET2 as
the reference method and Ridascreen SET Total as an
alternative method. Moreover, when results showed dis-
crepancies, the EU-RL for CPS in-house confirmatory
detection method (Lapeyre et al. 1987, 1988) was per-
formed. The relative accuracy, specificity and sensitivity
values were calculated according to the qualitative proce-
dure of the EN ISO 16140 Standard’s validation protocol.
Extraction of staphylococcal enterotoxins
All samples were prepared as previously described
(Hennekinne et al. 2007). Briefly, 25 g of cheese were
mixed in 40 ml of distilled water at 38C, using an Ultra
Turraxsample homogenizer. The sample was shaken at
room temperature for at least 30 min. The pH of the
slurry was adjusted to pH 3Æ5 ± 0Æ5 with 5 mol l)1 HCl
(Merck, Darmstadt, Germany) to precipitate caseins and
the sample was then centrifuged at 10 000 g at 4C for
15 min. The aqueous supernatant was adjusted to pH
7Æ3 ± 0Æ3 with 5 mol l)1 NaOH (Merck) and centrifuged
as above. The supernatant was filtered through glass wool
and concentrated on a dialysis membrane with MWCO of
6–8000 Da (Spectrum Laboratories Inc., Rancho Domin-
guez, CA, USA) against 30% polyethylene glycol 20 000
(Merck) overnight at 4C. The concentrated protein
extract was recovered and adjusted to a final weight of 5 g
using phosphate buffered saline (PBS: 145 mmol l)1 ⁄
10 mmol l)1 NaCl ⁄ Na2HPO4, pH = 7Æ3 ± 0Æ2).
Detection of staphylococcal enterotoxins
Vidas SET2 kit (used as the reference method in this
study). As part of the validation procedure, this method
has been described in detail (Hennekinne et al. 2007).
The Vidas SET2 detection is based on an ELFA test.
Briefly, the Vidas SET2 is a rapid and fully automated
kit that detects the staphylococcal enterotoxins types A to
E, without differentiating among them, by using a tip
coated with specific antibodies for SEA, SEB, SECs, SED
and SEE. An immune complex is formed between (i) the
coated antibodies, (ii) the toxins in the concentrated
extract and (iii) the anti-SE antibodies conjugated with
alkaline phosphatase. All reagents are included in the
wells of the testing strip. Then, 500 ll of the concentrated
protein extract to be tested or 500 ll of the controls
(positive C1 and negative C2) are distributed on the
strips and incubated in the dedicated automated analyser.
The automated analyser carries out a fluorescence mea-
surement. The threshold value is set to 0Æ13. The sample
is deemed to contain no SEs or SEs at a concentration
below the detection limit if the test value (TV) is less
469
SEA to SEE detection in cheeses A. Ostyn et al.

than the threshold value. The sample is considered to be
contaminated by SEs if the TV is greater or equal to the
threshold value.
Ridascreen SET Total kit (used as an alternative method
in this study). The Ridascreen SET Total is a sandwich-
type enzyme immunoassay (ELISA) for detecting the
combined staphylococcal enterotoxins types SEA to SEE.
The surface of a microtitre plate is coated with specific,
purified antibodies which can bind the enterotoxins
contained in a food sample. By adding specific anti-
bodies against the toxins, a sandwich complex is formed
(antibody–antigen–antibody complex). The presence of
enterotoxins is revealed by adding an enzyme sub-
strate ⁄ chromogen solution containing tetramethylbenzi-
dine. A blue colour indicates the presence of
staphylococcal enterotoxins in the sample. After addition
of a sulphuric acid solution, which leads to a colour
change from blue to yellow, the presence of SEs is deter-
mined using a spectrophotometer at a double wavelength
of 450 ⁄ 630 nm. The threshold value is calculated by add-
ing 0Æ15 to the negative control value. The sample is
deemed to contain no SEs or SEs at a concentration
below the detection limit if the absorbance test value is
less than the threshold value. The sample is considered to
be contaminated by SEs if the absorbance test value is
greater or equal to the threshold value.
EU-RL for CPS in-house confirmatory method performed in
case of discrepancies in results. Differential detection and
characterization of staphylococcal enterotoxins were per-
formed by a quantitative indirect sandwich-type ELISA
developed in our laboratory. Specific monoclonal anti-
bodies (Mabs) (Lapeyre et al. 1987) are used as coating
antibodies and rabbit polyclonal antibodies as probing
antibodies. After adding goat anti-rabbit immuno-
globulins coupled to horseradish peroxidase, the presence
of staphylococcal enterotoxins is revealed by a sub-
strate ⁄ chromogen solution and a colorimetric measure-
ment. This method has been described in detail by
Hennekinne et al. (2007).
Intra-laboratory validation procedure
Characterization of the Ridascreen SET Total detection kit
and comparison with the Vidas SET2 kit. The EN ISO
16140 Standard was used for the intra-laboratory valida-
tion of the Ridascreen SET Total kit for the detection of
staphylococcal enterotoxins SEA to SEE in cheese.
Before conducting the validation procedure on natu-
rally contaminated and non-contaminated cheeses, a pre-
liminary study was performed. The relative limits of
detection of the Ridascreen SET Total and the Vidas
SET2 detection kits were studied by using (i) phosphate
buffer solutions spiked with SE types A, B, C2, D and E
(Toxin Technology, Sarasota, FL, USA) at increasing con-
centrations (0, 0Æ031, 0Æ062, 0Æ125, 0Æ250, 0Æ500 and
1Æ000 ng g)1) and (ii) non-contaminated cheeses spiked
with SEA (at 0Æ500 ng g)1), SEB (at 0Æ500 ng g)1), SEC2
(at 2Æ000 ng g)1), SED (at 0Æ500 ng g)1) or SEE (at
2Æ000 ng g)1).
Determination of performance parameters. In this study,
the Vidas SET2 detection kit was used as the reference
method and the Ridascreen SET Total was considered as
the alternative method to determine statistical parameters
described in the EN ISO 16140 Standard. For this pur-
pose, parameters such as relative accuracy (AC), sensitiv-
ity (SE) and specificity (SP) were calculated with their
confidence intervals (Table 1).
Results
Preliminary study: sensitivity and specificity determina-
tion in buffer solutions and spiked cheeses
Results of the determination of the sensitivity of the Rida-
screen SET Total and the Vidas SET2 detection kits are
shown in Table 2. The results on sensitivity showed that
the relative limits of detection for SEA and SEE could be
considered as equivalent for both detection methods.
Regarding SEB, the Vidas SET2 detected lower entero-
toxin amounts than the Ridascreen SET Total. In con-
trast, the Ridascreen SET Total detected lower amounts
of SEC2 and SED than those detected with the Vidas
SET2 detection kit. Regarding the results of spiked
Table 1 Accuracy parameters according to the EN ISO 16140
Standard
Reference method
Positive Negative
Alternative method
Positive Positive Positive
agreement (PA) deviation (PD)
Negative Negative Negative
deviation (ND) agreement (NA)
Relative accuracy (AC) AC = (PA + NA) ⁄ N*
Relative specificity (SP) SP = (NA ⁄ N-)
Relative sensitivity (SE) SE = (PA ⁄ N+)
*
N, total number of samples (NA + PA + PD + ND).

N-, number of negative samples according to the reference method
(NA + PD).

N+, number of positive samples according to the reference method
(PA + ND).

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470 Letters in Applied Microbiology 52, 468–474 ª 2011 The Society for Applied Microbiology
A. Ostyn et al.
Table 2 Evaluation of the sensitivity of the Ridascreen SET Total and the Vidas SET2 detection kits
Buffer solution
 
Vidas SET2
(ng ml)1)
SEA £0Æ031
SEB £0Æ031
SEC2 0Æ250
SED 0Æ125
SEE £0Æ031
DC, dialysis concentration.
cheeses, the sensitivity for the five SE toxins was similar
for both kits.
Additional experiments were conducted to determine
the specificity of both Vidas SET2 and Ridascreen SET
Total kits of a non-contaminated buffer solution and a
non-spiked cheese. SEA to SEE were not detected in these
samples using Vidas SET2 and Ridascreen SET Total
kits (data not shown).
Intra-laboratory validation procedure: detection of staph-
ylococcal enterotoxins in cheeses
Among the 65 samples of cheeses tested, 30 samples were
clearly positive and 33 negative, whatever the method
used. Two out of 65 results showed discrepancies
(Table 3).
Determination of the relative accuracy, specificity and
sensitivity parameters
Table 4 presents the numbers of positive and negative
agreements, as well as positive and negative deviations
used to determine statistical parameters.
The results of the comparison of the SEs detection for
both kits gave the same value of 97% for relative accu-
racy, sensitivity and specificity, with confidence intervals
of )4%, )5% and )5%, respectively. As the number of
results with discrepancies was lower than six, no statistical
analysis could be conducted to conclude on the statistical
difference between the two methods. Given the high val-
ues of the relative accuracy, sensitivity and specificity,
both methods could be considered as equivalent in terms
of detecting SEA to SEE in naturally contaminated
cheeses.
The in-house confirmatory method was performed in
the two cases showing discrepancies between the Vidas
SET2 and Ridascreen SET Total detection kits. In the
first case, sample 21, SEs were detected by the Rida-
screen SET Total kit but not by the Vidas SET2 kit
(Table 3). However, the TV (0Æ12) for the Vidas SET2
ª 2011 The Authors
Letters in Applied Microbiology 52, 468–474 ª 2011 The Society for Applied Microbiology
SEA to SEE detection in cheeses
Spiked cheese
Ridascreen SET DC and Vidas DC and Ridascreen
total (ng ml)1) SET2 (ng ml)1) SET total (ng g)1)
£0Æ031 £0Æ012 £0Æ012
£0Æ031 £0Æ012 0Æ025
£0Æ031 0Æ100 0Æ025
0Æ062 0Æ125 0Æ050
£0Æ031 0Æ050 0Æ050
was very close to the threshold (0Æ13). The difference
between the two kits could be attributed to differences in
the limit of detection (Table 2). Furthermore, determina-
tion of the sensitivity of the two detection kits showed
that the Ridascreen SET Total kit was able to detect
lower amounts of SEC2 and SED than the Vidas SET2
kit. Accordingly, the in-house confirmatory method
detected SEC and SED toxins, although they were not
quantified. In the second case, sample 45, SEs were
detected with Vidas SET2 kit but not with Ridascreen
SET Total kit (Table 3). This discrepancy may be linked
to the presence of alkaline phosphatase in the sample
interfering with the Vidas SET2 test. After heat treat-
ment of the extract (two minutes at 80C), the result was
negative with Vidas SET2, suggesting the presence of
heat-sensitive alkaline phosphatase in the extract. More-
over, the EU-RL for CPS in-house confirmatory quantita-
tive ELISA method did not detect any of the SEA to SEE
toxins. In both cases (samples 21 and 45), the in-house
confirmatory method confirmed the results obtained with
the Ridascreen SET Total kit.
The comparison of the detection using Ridascreen
SET Total and the Vidas SET2 together with the in-
house confirmatory method did not show any deviations.
Thus, parameters such as AC, SE and SP were all equal to
100%. Therefore the Ridascreen SET Total kit can be
considered to be reliable for detecting SEA to SEE in
dairy products.
Discussion
European Regulation (EC) No. 2073 ⁄ 2005 (Anon. 2005)
modified by the Regulation (EC) No. 1441 ⁄ 2007 (Anon.
2007) requires enumeration of CPS in milk products dur-
ing the manufacturing process at the processing step
where the number of staphylococci is expected to be the
highest. If the CPS counts are higher than 105 CFU g)1,
SEs must be screened for. Milk products are regularly
involved in food-borne diseases due to the occurrence of
CPS in raw milk and ⁄ or in milk products due to cross-
471
SEA to SEE detection in cheeses A. Ostyn et al.

Table 3 Detection of staphylococcal enterotoxin types SEA to SEE in cheese samples

Sample Origin of DC and DC and Ridascreen DC and in-house

number milk Vidas SET2 SET Total confirmatory method Interpretation

1 Cow + + P.A.
2 + + P.A.
3 ) ) N.A.
4 ) ) N.A.
5 ) ) N.A.
6 + + P.A.
7 ) ) N.A.
8 ) ) N.A.
9 ) ) N.A.
10 + + P.A.
11 + + P.A.
12 + + P.A.
13 + + P.A.
14 + + P.A.
15 + + P.A.
16 + + P.A.
17 + + P.A.
18 + + P.A.
19 + + P.A.
20 + + P.A.
21 ) + + P.D.
22 ) ) N.A.
23 + + P.A.
24 + + P.A.
25 + + P.A.
26 ) ) N.A.
27 + + P.A.
28 Sheep + + P.A.
29 + + P.A.
30 ) ) N.A.
31 + + P.A.
32 + + P.A.
33 ) ) N.A.
34 Goat ) ) N.A.
35 ) ) N.A.
36 ) ) N.A.
37 ) ) N.A.
38 ) ) N.A.
39 ) ) N.A.
40 ) ) N.A.
41 ) ) N.A.
42 ) ) N.A.
43 + + P.A.
44 + + P.A.
45 Not specified + ) ) N.D.
46 ) ) N.A.
47 ) ) N.A.
48 ) ) N.A.
49 ) ) N.A.
50 ) ) N.A.
51 ) ) N.A.
52 ) ) N.A.
53 ) ) N.A.
54 ) ) N.A.
55 ) ) N.A.

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472 Letters in Applied Microbiology 52, 468–474 ª 2011 The Society for Applied Microbiology
A. Ostyn et al. SEA to SEE detection in cheeses

Table 3 (Continued)

Sample Origin of DC and DC and Ridascreen DC and in-house

number milk Vidas SET2 SET Total confirmatory method Interpretation

56 ) ) N.A.
57 ) ) N.A.
58 ) ) N.A.
59 ) ) N.A.
60 + + P.A.
61 + + P.A.
62 + + P.A.
63 + + P.A.
64 + + P.A.
65 + + P.A.

DC, dialysis concentration; P.A., positive agreement; N.A., negative agreement; P.D., positive deviation; N.D., SEA to SEE not detected.

Table 4 Summary of the comparative results on the Vidas SET2 and
Ridascreen SET Total detection kits
Vidas SET2
Positive
Ridascreen SET total
Positive 30
Negative 1 33
contamination during processing. Thus, to protect con-
sumers, there is a crucial need of specific and sensitive
methods to detect low amounts of staphylococcal entero-
toxins in these foods.
For this purpose, the EU-RL for CPS (Anon. 2006)
develops and characterizes methods for the detection of
staphylococcal enterotoxin types SEA to SEE in dairy
products in compliance with European Regulation (EC)
No. 2073 ⁄ 2005. As part of evaluating a screening method
for dairy products, the EU-RL for CPS performed an
intra-laboratory study based on the EN ISO 16140 Stan-
dard, coupling extraction and subsequent sample concen-
tration by dialysis with the Ridascreen SET Total
detection kit. The results of this study show that the
Ridascreen SET Total kit is sensitive and specific enough
to detect SEA to SEE in milk products. Thus, NRLs for
CPS can now use, after an extraction step followed by
dialysis concentration, Vidas SET2 or Ridascreen SET
Total kits to perform official controls for detecting SEA
to SEE in milk and milk-based products (Anon. 2010b).
The outcome of an inter-laboratory validation trial is in
progress to fully validate the Ridascreen SET Total as a
screening method to detect SEs in food, including milk
and milk products.
Methods based on techniques other than immunoas-
says nevertheless need to be considered to guarantee
food safety with regard to SEs. Alternative methods
include molecular-based techniques, such as the poly-
merase chain reaction (PCR), which are able to detect
se-encoding genes in strains of Staph. aureus isolated
from contaminated foods. However, this methodology
has two major limitations: first, staphylococcal strains
Negative must be isolated from the incriminated food and, sec-
ond, the results determine whether se-encoding genes
1 are present or absent, but cannot attest to actual se-gene
expression in food. While the PCR approach usefully
supplements immunology-based methods, it cannot be
used as a unique tool to detect SEs. Another very prom-
ising new approach is based on analytical chemistry and
consists in quantifying staphylococcal enterotoxins by
liquid chromatography coupled with tandem mass spec-
trometry. This type of methodology has been developed
for detecting and quantifying SEA in standard solutions
and in a few matrices, such as dessert foods and cheese
(Dupuis et al. 2008; Hennekinne et al. 2009). It is one
the most sensitive techniques currently available and it
provides specific, rapid and reliable analytical results.
However, because this method is expensive, requires a
larger scale of food and has not yet been extended to
SEs other than SEA, it is a tool that is most suited to
qualified laboratories that characterize and investigate
SFPOs.
In conclusion, several approaches are probably needed
to fully characterize a contamination event in food.
Screening methods are a very important tool for official
controls as they can easily be performed on a large scale.
The intra-laboratory validation performed here in accor-
dance with the EN ISO 16140 Standard yielded satisfac-
tory results. This study showed that the methodology
developed here and based on sample concentration by
dialysis coupled with detection by Ridascreen SET Total
kit is easy to use, sensitive and specific enough to detect
SEA to SEE in milk and milk products. This alternative
to the VidasSET2 kit may be useful for official controls,
pending confirmation by inter-laboratory test results.

ª 2011 The Authors

Letters in Applied Microbiology 52, 468–474 ª 2011 The Society for Applied Microbiology 473
SEA to SEE detection in cheeses
Acknowledgements
This work was partly conducted under the auspices of the
European Union-Reference Laboratory for coagulase posi-
tive staphylococci including Staphylococcus aureus, funded
by the Health and Consumer Protection Directorate Gen-
eral (DG SANCO) of the European Commission.
Authors would like to acknowledge the bacterial toxins
team of the NRL and EU-RL for CPS for its participation
in this study, and the French laboratories involved in offi-
cial controls for CPS, ACTILAIT (La Roche-sur-Foron,
France) and the Lactic Acid Bacteria and Opportunistic
Pathogens laboratory (UBLO) of the French National
Institute for Agricultural Research (INRA) (Jouy-en-Josas,
France) for providing some of the cheese samples used in
this study.
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ª 2011 The Authors