First report of enterotoxigenic Staphylococcus argenteus as a foodborne pathogen.

Staphylococcal enterotoxins preformed in food are the causative agents of staphylococcal food poisoning outbreaks (SFPO). In this study we characterised in depth two coagulase-positive non-pigmented staphylococci involved in two independent outbreaks that occurred in France. While indistinguishable from Staphylococcus aureus using PCR methods and growth phenotype comparisons, both isolates were identified as Staphylococcus argenteus by whole genome sequencing. The genomes were analysed for the presence of enterotoxin genes, whose expression was determined in laboratory medium and, for the first time, in artificially-contaminated milk samples by using liquid chromatography-mass spectrometry and ELISA methods. The concentration measured for the SEB toxin in milk (0.67 ng/ml) was comparable to concentrations reported for other types of enterotoxins behind SFPO. From a collection of publicly available genomes, we performed an unprecedented systematic investigation of the enterotoxin gene set of S. argenteus, including variants and pseudogenes. The most prevalent genes were sex, followed by sel26, sel27 and sey. The egc cluster was less frequent and most of the time carried a dysfunctional seg gene. Our results shed light on the enterotoxigenic properties of S. argenteus, and emphasize the importance in monitoring of S. argenteus as an emerging foodborne pathogen.

Insight Into the Distribution of Staphylococci and Their Enterotoxins in Cheeses Under Natural Conditions.

Staphylococcal food poisoning outbreaks are a major cause of food-borne illness in the European Union and their notification has been mandatory since 2005. Criteria for the enumeration of coagulase-positive Staphylococci (CPS) and the detection of staphylococcal enterotoxins (SEs) in cheese have been set down in Commission Regulation EC 2073/2005. Currently, few information are available about the distribution of SEs in naturally contaminated cheeses, including raw-milk and artisanal dairy products. The aim of this study was therefore to investigate at both the CPS enumeration and the succession of the enterotoxigenic Staphylococcus aureus and produced enterotoxins levels on the rind and the core of a raw-milk semi-hard cheese, produced on farm. The study has been conducted in three steps: (I) seven wheels at different time of ripening where tested for the presence of SEs. (II) from each wheel, four portions were subsequently sampled from four different areas (peripheral rind, central rind, peripheral core and central core). (III) two cheese wheels, characterized by the highest and lowest CPS numbers and SEs quantification, based on the second step of the study, were further analyzed. A significant difference has been observed in the distribution of CPS and SEs in the four areas sampled, irrespectively of the batch and the time of ripening. The results of this study provided a set of previously unknown information on the influence of natural conditions on the distribution of CPS and SEs thereof in the cheese matrix, filling a gap in the understanding of SEs biosynthesis process.

Certified reference materials for testing of the presence/absence of Staphylococcus aureus enterotoxin A (SEA) in cheese.

Staphylococcal enterotoxins (SEs) account for a substantial number of food-poisoning outbreaks. European legislation (Commission Regulation 1441/2007) stipulates the reference procedure for SE analysis in milk and dairy products, which is based on extraction, dialysis concentration and immunochemical detection using one of two approved assays (VIDAS(®) SET2, Ridascreen(®) SET Total). However, certified reference materials (CRMs) are lacking to support laboratories in performing reliable detection of Staphylococcus aureus enterotoxin A (SEA) in relevant matrices at sub-nanogram per gram levels. The certification of a set of three reference materials (blank and two SEA-containing materials) for testing of the presence/absence of SEA in cheese is described. The reference procedure was applied in an intercomparison with 15 laboratories, and results were reported in a qualitative manner (presence or absence of SEA in the sample). No false-negative or false-positive results were obtained. The certified values were stated as diagnostic specificity (blank material) or diagnostic sensitivity (SEA-containing materials) and were 100 % in all cases. Stability studies demonstrated suitable material stability when stored cooled or frozen. An in-house study on the recovery of SEA in the cheese materials using a double-sandwich enzyme-linked immunosorbent assay (ELISA) revealed comparable recovery values of around 45 % at the two spiking levels and in both the SEA-containing CRMs as well as blank CRM freshly spiked prior to analysis. The values were also comparable over time and among different analysts. The materials provide valuable support to laboratories for method validation and method performance verification and will increase the reliability of measuring SEA in cheese.

Review Over a 3-Year Period of European Union Proficiency Tests for Detection of Staphylococcal Enterotoxins in Food Matrices.

Staphylococcal food poisoning outbreaks are a major cause of foodborne illnesses in Europe and their notifications have been mandatory since 2005. Even though the European regulation on microbiological criteria for food defines a criterion on staphylococcal enterotoxin (SE) only in cheese and dairy products, European Food Safety Authority (EFSA) data reported that various types of food matrices are involved in staphylococcal food poisoning outbreaks. The European Screening Method (ESM) of European Union Reference Laboratory for Coagulase Positive Staphylococci (EURL CPS) was validated in 2011 for SE detection in food matrices and is currently the official method used for screening purposes in Europe. In this context, EURLCPS is annually organizing Inter-Laboratory Proficiency Testing Trials (ILPT) to evaluate the competency of the European countries' National Reference Laboratories (NRLs) to analyse SE content in food matrices. A total of 31 NRLs representing 93% of European countries participated in these ILPTs. Eight food matrices were used for ILPT over the period 2013-2015, including cheese, freeze-dried cheese, tuna, mackerel, roasted chicken, ready-to-eat food, milk, and pastry. Food samples were spiked with four SE types (i.e., SEA, SEC, SED, and SEE) at various concentrations. Homogeneity and stability studies showed that ILPT samples were both homogeneous and stable. The analysis of results obtained by participants for a total of 155 blank and 620 contaminated samples allowed for evaluation of trueness (>98%) and specificity (100%) of ESM. Further to the validation study of ESM carried out in 2011, these three ILPTs allowed for the assessment of the proficiency of the NRL network and the performance of ESM on a large variety of food matrices and samples. The ILPT design presented here will be helpful for the organization of ILPT on SE detection by NRLs or other expert laboratories.