CERTIFICATION REPORT Certified reference materials for testing of the presence/absence of Staphylococcus aureus enterotoxin A (SEA) in cheese: IRMM-359a-c

This report describes the preparation of three cheese powder matrix reference materials (IRMM-359a-c) and their certification for testing of the presence/absence of Staphylococcus aureus enterotoxin A (SEA). Raw milk cheese was decrusted, cut into cubes, chopped in a kitchen-type food processor for a short time, freeze-dried, cryogenically milled, and mixed (blank material IRMM-359a). Moreover, a second portion of raw milk cheese was decrusted and cut into cubes. After addition of water and spiking with a solution of SEA, the sample was homogenised using a high-speed grinder (Ultra-Turrax). The cheese slurry was freeze-dried, cryogenically milled and mixed with blank cheese powder to obtain the two SEA-containing materials at SEA target levels of 0.1 and 0.25 ng/g cheese, respectively (IRMM-359b, IRMM-359c). Between unit-homogeneity was quantified and stability during dispatch and storage were assessed in accordance with ISO Guide 35:2006. The minimum sample intake is 15.1 g cheese powder (representing 25 g of cheese after reconstitution) per replicate analysis (n=5), as stipulated in Commission Regulation 1441/2007, and therefore no dedicated study on the minimum sample intake was performed. The reference material was characterised in an interlaboratory comparison of laboratories of demonstrated competence and adhering to ISO/IEC 17025 and using the European Screening Method with the VIDAS SET2 and the Ridascreen SET Total for detection (further on named ESM/VIDAS and ESM/Ridascreen, respectively) [4]. Technically invalid results were removed, but no outlier was eliminated on statistical grounds only. Certified values are reported as probability of detection and expressed as either diagnostic specificity (ratio of true negatives divided by the sum of true negatives and false positives) for the blank material, or diagnostic sensitivity (ration of true positives divided by the sum of true positives and false negatives) for the SEA-containing materials. Uncertainties for homogeneity and stability were estimated, but not used for an uncertainty budget due to the nature of the certified values (presence/absence certification). Instead, the certified values are expressed as intervals with a 95% level of confidence. The preparation and processing of the material, homogeneity and stability studies, and the characterisation are described hereafter and the results are discussed.JRC.D.2-Standards for Innovation and sustainable Developmen

Staphylococcus aureus virulence and metabolism are dramatically affected by Lactococcus lactis in cheese matrix

International audienceIn complex environments such as cheeses, the lack of relevant information on the physiology and virulence expression of pathogenic bacteria and the impact of endogenous microbiota has hindered progress in risk assessment and control. Here, we investigated the behaviour of Staphylococcus aureus, a major foodborne pathogen, in a cheese matrix, either alone or in the presence of Lactococcus lactis, as a dominant species of cheese ecosystems. The dynamics of S. aureus was explored in situ by coupling a microbiological and, for the first time, a transcriptomic approach. Lactococcus lactis affected the carbohydrate and nitrogen metabolisms and the stress response of S. aureus by acidifying, proteolysing and decreasing the redox potential of the cheese matrix. Enterotoxin expression was positively or negatively modulated by both L. lactis and the cheese matrix itself, depending on the enterotoxin type. Among the main enterotoxins involved in staphylococcal food poisoning, sea expression was slightly favoured in the presence of L. lactis, whereas a strong repression of sec4 was observed in cheese matrix, even in the absence of L. lactis, and correlated with a reduced saeRS expression. Remarkably, the agr system was downregulated by the presence of L. lactis, in part because of the decrease in pH. This study highlights the intimate link between environment, metabolism and virulence, as illustrated by the influence of the cheese matrix context, including the presence of L. lactis, on two major virulence regulators, the agr system and saeRS

How Should Staphylococcal Food Poisoning Outbreaks Be Characterized?

Staphylococcal food poisoning is one of the most common food-borne diseases and results from the ingestion of staphylococcal enterotoxins (SEs) preformed in food by enterotoxigenic strains of Staphylococcus aureus. To date, more than 20 SEs have been described: SEA to SElV. All SEs have superantigenic activity whereas only a few have been proved to be emetic, representing a potential hazard for consumers. Characterization of staphylococcal food poisoning outbreaks (SFPOs) has considerably progressed compared to 80 years ago, when staphylococci were simply enumerated and only five enterotoxins were known for qualitative detection. Today, SFPOs can be characterized by a number of approaches, such as the identification of S. aureus biovars, PCR and RT-PCR methods to identify the se genes involved, immunodetection of specific SEs, and absolute quantification by mass spectrometry. An integrated gene-to-protein approach for characterizing staphylococcal food poisoning is advocated

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

Innovative approaches to improve staphylococcal food poisoning characterization

Staphylococcal food poisoning is one of the most common food-borne diseases resulting from the ingestion of staphylococcal enterotoxins (SEs) preformed in food by enterotoxigenic strains of coagulase-positive staphylococci, mainly Staphylococcus aureus. To date, more than twenty SEs have been described: SEA to SElV. Each of them share superantigenic activity whereas only few of them have been proved to be emetic and thus represent a potentiel hazard for consumers. Characterisation of staphylococcal food poisoning outbreaks are dependent of various parameters such as isolation of coagulase positive staphylococci and detection of staphylococcal enterotoxins. However, various drawbacks such as heat treatment sensitivity of staphylococci or availability of antibodies against all staphylococcal enterotoxins lead to false diagnosis or to a lack of characterisation of food poisoning outbreaks. Thus, in order to characterise and elucidate staphylococcal food poisoning, we developed an integrated approach from gene to protein using the combination of PCR-based tools, ELISA tests and quantitative MS.Les toxi-infections alimentaires collectives staphylococciques représentent ces dernières années en France la seconde cause de toxi-infections alimentaires bactériennes. Ces dernières sont dues à l'ingestion de toxines préformées dans les aliments : les entérotoxines. Actuellement, une vingtaine d'entérotoxines (SEA àSElV) ont été décrites dans la littérature ; parmi celles-ci, toutes possédent une activité superantigéniques mais seules quelques unes possèdent une activité émétique prouvée et présentent donc un risque sanitaire pour le consommateur. Deux problèmes majeurs se posent pour confirmer le diagnostic des épisodes toxiques à staphylocoques. D'une part, les staphylocoques sont sensibles aux traitements thermiques appliqués aux aliments alors que les entérotoxines ne le sont pas, et d'autre part les méthodes de détection des entérotoxines staphylococciques actuellement disponibles ne couvrent pas l'ensemble des entérotoxines décrites à ce jour. Ainsi, et afin de proposer une alternative innovante aux tests immuno-enzymatiques utilisés pour la recherche des entérotoxines staphylococciques dans les matrices alimentaires, nous avons mis en place une démarche intégrée « du gène à la protéine » utilisant des outils de biologie moléculaire, d'immunochimie et de physisco-chimie des protéines pour caractériser, comprendre et élucider les toxi-infections alimentaires collectives à staphylocoques

First Proficiency Testing To Evaluate the Ability of European Union National Reference Laboratories To Detect Staphylococcal Enterotoxins in Milk Products

The European Commission has designed a network of European Union-National Reference Laboratories (EU-NRLs), coordinated by a Community Reference Laboratory (CRL), for control of hygiene of milk and milk products (Council Directive 92/46/ECC). As a common contaminant of milk and milk products such as cheese, staphylococcal enterotoxins are often involved in human outbreaks and should be monitored regularly. The main tasks of the EU-CRLs were to select and transfer to the EU-NRLs a reference method for detection of enterotoxins, and to set up proficiency testing to evaluate the competency of the European laboratory network. The first interlaboratory exercise was performed on samples of freeze-dried cheese inoculated with 2 levels of staphylococcal enterotoxins (0.1 and 0.25 ng/g) and on an uninoculated control. These levels were chosen considering the EU regulation for staphylococcal enterotoxins in milk and milk products and the limit of detection of the enzyme-linked immunosorbent assay test recommended in the reference method. The trial was conducted according to the recommendations of ISO Guide 43. Results produced by laboratories were compiled and compared through statistical analysis. Except for data from 2 laboratories for the uninoculated control and cheese inoculated at 0.1 ng/g, all laboratories produced satisfactory results, showing the ability of the EU-NRL network to monitor the enterotoxin contaminant

Role of milk and milk products in the spread of methicillin‐resistant Staphylococcus aureus in the dairy production chain

International audienceMilk and milk products can harbor a multiple varieties of microorganisms. Therefore, they can be an important source of foodborne pathogens, including multidrug-resistant bacteria. Methicillin-resistant Staphylococcus aureus (MRSA) causes a wide spectrum of infections both in animals and humans. Over the last two decades, the presence of MRSA in foods and food-producing animals, including milk and milk products, has been frequently reported worldwide, raising public health concerns. In order to monitor and prevent foodborne MRSA contamination, it is necessary to understand their sources, the pheno/genotypic characteristics of the strains, and their transmission dynamics. In this review, studies conducted worldwide were summarized in order to assess the prevalence and diversity of MRSA circulating in milk and milk products. The risk factors for the occurrence of MRSA in milk and milk products were also discussed with preventive and control measures to avoid MRSA contamination in the dairy food chain

Dose-response Modelling of Staphylococcal Enterotoxins Using Outbreak Data

AbstractStaphylococcal food poisoning (SFP) is one of the most common food-borne diseases and results from the ingestion of staphylococcal enterotoxins (SEs). Yet, small amount of data are available for establishing a dose response. The objective of this work was to build a dose response relation based on the systematic investigations carried out during recent years in France. Over the period 2010-2014, more than 60 SFP outbreaks involving SEs, mainly from France, were microbiologically investigated. The enterotoxins were characterized as well as quantified. Attack rates, appearance times and natures of symptoms collected during epidemiological investigations were related to microbiological data. The outbreaks collected focused on enterotoxins SEA, SEB, SEC, and SED. Distribution of appearance times of symptoms and their natures were not influenced by the type of enterotoxins. The US EPA benchmark dose (BMD) methodology was then used to establish dose response. Attack rates of SFP outbreaks were modelled as a function of ingested doses and a BMD have been estimated for SEA

Organization and ELISA-Based Results of the First Proficiency Testing to Evaluate the Ability of European Union Laboratories to Detect Staphylococcal Enterotoxin Type B (SEB) in Buffer and Milk

The aim of this work was to organize the first proficiency test (PT) dedicated to staphylococcal enterotoxin B (SEB) detection in milk and buffer solutions. This paper describes the organization of the PT trial according to EN ISO 17043 requirements. Characterization of the SEB stock solution was performed using SDS-PAGE and SE-specific ELISA, and amino acid analysis was used to assign its protein concentration. The solution was then used to prepare six PT materials (four milk and two buffer batches) at a ng/g toxin level, which included one blank and one SEA-containing milk as specificity control. Suitable material homogeneity and stability were assessed using screening and quantitative ELISAs. Among the methods used by the participants, ELISA-based methods demonstrated their efficiency for the detection of SEB in both simple and complex matrices. The results serve as a basis for further improving the detection capabilities in expert laboratories and can therefore be considered as a contribution to biopreparedness