Livia Schwendimann

D Merda

Thomas Berger

Sarah Denayer

C Feraudet Tarisse

A J Kläui

Sabine Messio

Michel-Yves Y Mistou

Yacine Nia

Jacques-Antoine A Hennekinne

Hans Ulrich Graber

Ivana Ivanovic

Staphylococcal enterotoxin gene cluster: prediction of enterotoxin (SEG and SEI) production and of the source of food poisoning based on vSaβ typing 1 (Paper I)

Keywords: Tier kann Staphylococcus aureus in Lebensmitteln auch verschiedene Enterotoxine produzieren. Diese Enterotoxine können Erbrechen und Durchfall verursachen. Sind mehrere Personen betroffen, kommt es zu sogenannten Staphylococcal Food Poisoning Outbreaks (SFPO). Aus verschiedenen Gründen sind viele dieser SFPOs nur schwach belegt. Ein Grund dafür ist, dass von den 26 bekannten Enterotoxinen derzeit nur die fünf klassischen mit kommerziell erhältlichen Kits analysiert werden können. Eine Untergruppe der nicht-klassischen Enterotoxine (seg, sei, sem, sen, seo und seu), werden durch denselben enterotoxin gene cluster konnten wir zeigen, dass die in vitro-Produktion von SEG und Beitrag zum besseren Queste enterotossine possono causare vomito e diarrea, spesso coinvolgendo molte persone Staphylococcus aureus, cheese production, scalding temperature, staphylococcal enterotoxin, cheese model

Beside the infection in human beings and animals, Staphylococcus aureus can produce different enterotoxins in food. These enterotoxins can cause vomiting and diarrhea, often involving many people and causing so called staphylococcal foodborne outbreaks (SFPO). To date a lot of SFPO are of weak evidence. One reason for this could be, that currently only five, so called classical enterotoxins, out of 26 known staphylococcal enterotoxins can be analyzed using commercially available kits. A subset of the non-classical enterotoxins (seg, sei, sem, sen, seo and seu) is located on the same enterotoxin gene cluster (egc) which is part of the S. aureus genomic island vSaβ. These enterotoxins have been described as likely being involved in SFPOs.

New tools such as whole genome sequencing and new methods for enterotoxin measurement are needed in order to be able to analyse the S. aureus strains and their enterotoxins and, consequently, being able to predict the enterotoxin production based on genome data. This was the main aim of this work.

In order to do so, the egc enterotoxins were studied at genome (seg, sei, sem, sen, seo, and seu), at mRNA (seg, sei, sem, sen, seo), and at protein level (SEG and SEI).

We demonstrate that the in vitro production of SEG and SEI can be predicted based on the vSaβ-type and the clonal complex (CC) of a strain. Furthermore, the vSaβ-type/CC enables the prediction of the source of an egc-positive SFPO strain (animal or human derived).

Enterotoxin production (SEG and SEI) under stress conditions was also tested, showing that common salt concentrations and temperature stress, mimicking conditions found in food production, seem not to affect the production of egc enterotoxins SEG and SEI.

With the present work a contribution can be given to a better understanding of the involvement of egc enterotoxins producing S. aureus strain in SFPOs. In addition, the results also give a first insight into the conditions and limits of egc enterotoxin production and a first indication in how they have the ability to be produced in food production and storage.

Résumé (French abstract)

Outre les infections qu'il peut causer chez l'homme et les animaux, le Staphylococcus aureus peut produire différentes entérotoxines dans les aliments. Ces entérotoxines peuvent provoquer des vomissements et des diarrhées, qui touchent souvent de nombreuses personnes et provoquent ce que l'on appelle des toxi-infections collectives (TIAC) à staphylocoques. À ce jour, de nombreux cas de TIAC sont peu patents, ceci pour de nombreuses raisons. L'une d'elles pourrait être qu'actuellement, seulement cinq entérotoxines, dites classiques, sur les 26 entérotoxines staphylococciques connues, peuvent être analysées à l'aide de kits disponibles dans le commerce. Une série d'entérotoxines non classiques (seg, sei, sem, sei, seo et seu) est située sur le même cluster (enterotoxin gene cluster, egc) qui fait partie de l'îlot génomique vSaβ de S. aureus. Ces entérotoxines ont été décrites comme pouvant être impliquées dans des toxi-infections alimentaires collectives à staphylocoques. C'est pourquoi de nouveaux outils, tels que le séquençage du génome complet (WGS) et de nouvelles méthodes de mesure des entérotoxines, sont nécessaires afin de pouvoir isoler et caractériser les souches de S. aureus et leurs entérotoxines et, par conséquent, de pouvoir prédire la production d'entérotoxines sur la base des données du génome. C'était le principal objectif de cette étude. Afin d'y parvenir, les entérotoxines egc ont été étudiées au niveau du génome (seg, sei, sem, sen, seo et seu), au niveau de l'expression de l'ARNm (seg, sei, sem, sen, seo) et au niveau des protéines (SEG et SEI). Avec les résultats, nous démontrons que la production in vitro de SEG et de SEI peut être prédite sur la base du type de vSaβ et du clonal complex (CC) d'une souche. De plus, le type de vSaβ/CC permet de prédire la source d'une souche TIAC positive à l'egc (d'origine animale ou humaine). La production d'entérotoxines (SEG et SEI) dans des conditions de stress a également été testée, montrant que les stress liés au NaCl et à la température, imitant les conditions rencontrées dans la production alimentaire, ne semblent pas affecter la production d'entérotoxines egc, SEG et SEI. La présente étude peut contribuer à mieux comprendre l'implication des souches de S. aureus produisant les énterotoxine egc dans les toxi-infections alimentaires collectives à staphylocoques. En outre, les résultats donnent également un premier aperçu des conditions et des limites de la production d'entérotoxines egc, ainsi qu'une première indication sur leur capacité à être produites dans la fabrication et le stockage des aliments.

Acknowledgement

First of all I would like to thank the two institutions (Agroscope and Anses) who made this PhD possible. The PhD would not have been possible without the personal engagement of different people of both institutions. Agroscope financing the PhD by giving me the possibility to do it as part of my regular employment and Anses giving the scientific, methodological support, as well as the link to the University Paris-Est. A very special thanks goes to Fabian Wahl and Hans-Peter Bachmann, opening me the path for doing a PhD, I'm extremely grateful for the opportunity you gave me.

A very special thanks goes to Michel-Yves Mistou, being the thesis director and giving every time the right inputs at the right moment. I would also like to thank Jacques-Antoine Hennekinne for being my supervisor at Anses, I'm glad I could rely on your deep knowledge on Staphylococcal enterotoxins.

A special thanks also goes to Hans-Ulrich Graber being my supervisor at Agroscope. I would like to thank you for the long scientific discussions, your continuous inputs that made me grew in the past three years. I really appreciate that.

I would also like to thank Thomas Berger for being my formal supervisor at Agroscope, for giving me extremely freedom in doing my work and the continuous support during the PhD time. Many thanks also goes to Yacine Nia for the perfect organization of my stays, lab and method preparation at Anses in Maisons-Alfort.

I would also like to thanks all the people of both laboratories where I had the pleasure to work.

From Anses a special thanks goes to Noémie Vingadassalon and Sabine Messio, without you the lab work would not have been possible. I'm also immense grateful to Déborah Mérda for the knowledge in genomics and bioinformatics, the big help for the results achieved and the great discussions on microbiology and live on-and beside work. From Agroscope a very special thanks go to Ivana Ivanovic for the perfect lab support and the great help in generating the results of my PhD. I'm also immensely grateful for the help of Anita Kläui, for her fabulous pre-studies on vSaβ, which were the perfect basis for my studies. I would also like to thank you for all your English proof reading and the very good exchanges. This PhD would not have been the same, without the grateful inputs of Yves Le Loir and Paola Cremonesi, as external members of the thesis committee you gave excellent help and perfect inputs to the thesis, thanks a lot. I would also like to thank Jenny Schelin and Georges Daube for being the reviewers of this thesis, I'm looking forward discussing with you at my PhD defence.

Many people contributed and made it possible that I got to the point where I'm now.

Firstly I would like to thanks my parents for giving me the opportunity to study and to pursue my dreams.

For my professional life I would like to thank Corinne Gantenbein, for being my first food microbiology teacher, without her I would not be so fascinated by these small organisms hidden in our foods. My thanks also go to Mathilde Hartmann Josefsen and Jeppe Boel, thanks to them I found the passion for food microbiology research and they gave me a unique experience at DTU in Denmark. Many thanks also go to Susanne Miescher Schwenninger, for being my master thesis supervisor and learning me how doing research really works and giving me the support and self confidence in pursuing the research path.

René Imhof for being like a mentor in my first years at Agroscope. Thanks for all your knowledge and discussion I could share with you.

A very special thank also goes to my closest friends: the "Chicas" who studied with me during the period at ZHAW. You gave me support whenever I needed it, telling me I will manage it, sometimes calling me to be like an emotional mammoth being able to survive everything. I would also like to thank Alicia, Carlotta and Desirée which I got to known at the lab at Agroscope and got closest friends on which I can always rely on. One very special person followed my life during the last twelve years, knowing me better than everybody else and also knowing the best how much I had the desire to do a PhD. Simon I would like to thank you for everything you did. For your constant support, the critical reading of my papers and the manuscript.

Last, but not less important I would like to thank Dani for your love and for taking my mind out of the PhD work whenever I spent time with you, allowing me the needed breaks and even more efficient returns to work.

"Our greatest weakness lies in giving up. The most certain way to succeed is always to try just one more time." -Thomas Edison

Introduction

Staphylococcus aureus is a widespread bacteria, present in different habitats like the human or animal skin. Due to contamination of raw material or/and through processing of food, the bacteria enters the food chain. Once there, under the right conditions (temperature, salt concentration, etc.), S. aureus can grow and often it produces enterotoxins. These enterotoxins are heat stable and whenever in a further processing step the food matrix is heated up, the bacteria will die but the enterotoxin will remain. In consequence by eating the food there is also an intake of enterotoxins. This is also the case in cheese production where the contamination happens mainly due to cow's mastitis. Before and in the first steps of cheese production S. aureus can grow and produce enterotoxins that will remain in cheese matrix.

Because these enterotoxins are superantigen and also have emetic activity, eating these enterotoxins causes vomiting and diarrhea. According to the European food safety authority in 2018 there were 114 foodborne outbreaks caused by S. aureus. Food involved seems mainly be mixed food, milk and meat products. 77 out of the 114 outbreaks are classified as weak evidence outbreaks, meaning that in these cases no causative agent could be detected in the food vehicle [START_REF] Efsa | Update of the technical specifications for harmonised reporting of food-borne outbreaks through the European Union reporting system in accordance with Directive 2003/99/EC[END_REF]. A reason for the high percentage of weak evidence outbreaks could be the fact that beside the so-called classical enterotoxins (SEA-SEE) which can be detected using commercially available kits, also non-classical enterotoxins are presumed to be involved.

For these ones there are no commercial kits available for their detection.

Background and motivation of the study

Agroscope, as Swiss center of excellence for agricultural research is affiliated with the Federal Office for Agriculture (FOAG). Agroscope makes an important contribution to a sustainable agriculture and food sector as well as to an intact environment, thereby contributing to an improved quality of life. In this frame Agroscope also has the duty to answer and provide data to the different stakeholders like the Federal Veterinary and Food Safety Office as well as the different agricultural and agricultural products supply chain supporters. Under this mandate Agroscope had to generate data in order to prove that in cheese production a scalding temperature of 52° C is sufficient to cause inactivation of all CPS present, and to prove that for this and higher temperatures no enterotoxin production takes place. These data were needed to show that cheese (semi-hard and hard cheese) analysis, on staphylococcal enterotoxin in alpine cheese manufactories, is not necessary to reach a sufficient food safety level.

The results of this study (see Appendix I), made with a lab cheese modelling system, aimed at reproducing the temperature curve in the peripheral zone of a real cheese, showed that enterotoxin formation occurred even at 56 °C and changed with the different scalding temperatures (52-56° C).

Interesting in this study was that the differences in enterotoxin formation were more associated with strain type rather than temperature. Based on these results it was decided that the mechanism of enterotoxin formation in cheese needs to be further analysed. Due to the fact that the topic is very wide and the knowledge on staphylococcal enterotoxin analysis is still very limited, a research partner needed to be found in order to fill this gap.

The perfect research partner was found with Anses and the "Laboratoire de sécurité des aliments de Maisons-Alfort" in the Staphylococcus, bacillus, clostridies (SBCL) unit. These food safety laboratories contribute to improve food quality and hygiene control through their expertise in analytical development. They provide support to public decision makers. The SBCL unit, performs studies on coagulase positive staphylococci (CPS), bacillus, clostridies involved in food poisoning outbreaks. The SBCL unit was nominated as EURL (European Union Reference Laboratories) for CPS and manages the NRL (National Reference Laboratory) network, conducts reference activities including development and validation of methods and organizes PT trails.

The collaboration between Agroscope and Anses was the starting point for this PhD. With a deep literature research, available methods and strains, specific research objective and research question were set up. These were the content of the present PhD thesis.

Overview

This thesis proceeds as follows. In Chapter 2, an overview of state-of-the-art research on S.

aureus and Staphylococcal enterotoxins is given. This includes an overview of the possible involvement and spread of the non-classical enterotoxins in foodborne outbreaks. This literature study was used to define on which staphylococcal enterotoxins the study should focus on. On the other side the literature research was also performed to check which methods are best used for the measurement of enterotoxin expression and production.

Based on the outcome of the literature study the objective and aim of the present work were defined (Chapter 3). Chapter 3.1 is giving an overview of the available and used strains and methods. In Chapter 5 the results of the PhD are presented under the form of research papers.

This chapter is divided into two subchapters: 5.1) dealing with the prediction and source tracking of staphylococcal enterotoxin production based on vSaβ and Clonal complex and 5.2)

where RNA expression studies were performed for strains exposed to stress conditions (salt concentration and temperature).

Finally, in Chapter 6 the results of the previous chapters are summarised and discussed, and in Chapter 7 conclusions are drawn.

Introduction (French)

Le Staphylococcus aureus est une bactérie très répandue, présente sur la peau autant des hommes que des animaux. Par contamination des matières premières et/ou à la suite de la transformation des aliments, la bactérie entre dans la chaîne alimentaire. Une fois qu'elle y est parvenue, si les conditions sont réunies (température, sel, etc.), S. aureus peut se développer et produire des entérotoxines. Ces entérotoxines résistent à la chaleur et à chaque traitement thermique ultérieur de la matrice alimentaire, la bactérie mourra, mais l'entérotoxine survivra.

Par conséquent, en mangeant cette nourriture, les entérotoxines sont ingérées. C'est également le cas dans la production de fromage, où la contamination est principalement due aux mammites des vaches. Avant et pendant les premières étapes de la production de fromage, S. aureus a la capacité de se développer et de produire des entérotoxines. A posteriori, même si le processus permet d'inactiver S. aureus, l'entérotoxine restera dans la matrice du fromage.

Étant donné que ces entérotoxines sont des super antigènes et ont également une activité émétique, leur consommation provoque des vomissements et des diarrhées. Selon l'Autorité européenne de sécurité des aliments, en 2018, 114 foyers de toxi-infections alimentaires ont été causés par S. aureus. Les aliments concernés semblent être principalement des aliments mélangés, des produits laitiers et carnés. 77 des 114 foyers sont classés comme des foyers non confirmés, ce qui signifie que dans ces cas, aucun agent causal n'a pu être détecté dans le support alimentaire [START_REF] Efsa | Update of the technical specifications for harmonised reporting of food-borne outbreaks through the European Union reporting system in accordance with Directive 2003/99/EC[END_REF]. Le pourcentage élevé de foyers non confirmés pourrait s'expliquer par le fait qu'outre les entérotoxines dites classiques (SEA-SEE) qui peuvent être détectées par les kits disponibles dans le commerce, des entérotoxines non classiques sont également susceptibles d'être impliquées. Or, il n'existe aucun kit commercial en mesure de les détecter.

Contexte et but de l'étude (French)

Agroscope, en tant que centre de compétence suisse pour la recherche agronomique, est rattaché à l'Office fédéral de l'agriculture (OFAG). Agroscope apporte une contribution importante à une agriculture et un secteur agro-alimentaire durables ainsi qu'à un environnement intact, permettant ainsi d'améliorer la qualité de vie. Dans ce cadre, Agroscope a également le devoir de répondre aux différentes parties prenantes comme l'Office fédéral de la sécurité alimentaire et des affaires vétérinaires ainsi qu'aux différents membres de la chaîne de distribution des produits agricoles et de leur fournir des informations. Dans le cadre de ce mandat, Agroscope doit générer des données afin de prouver que dans la production de fromage, une température de 52 °C est suffisante pour inactiver tous les SCP (staphylocoques à coagulase positive) présents, et qu'aucune entérotoxine n'est produite à cette température et à des températures plus élevées. Ces données étaient nécessaires pour montrer qu'il n'est pas nécessaire d'analyser les fromages (à pâte mi-dure et dure) pour détecter les entérotoxines staphylococciques dans les fromageries d'alpage.

Les résultats de cette étude (voir annexe I), réalisée avec un système de modélisation du fromage en laboratoire, visant à reproduire la courbe de température dans la zone périphérique d'une vraie meule de fromage, ont montré, contrairement aux attentes, que la formation d'entérotoxines pouvait se produire jusqu'à 56 °C et variait avec les différentes températures de fabrication (52-56 °C).

Il est intéressant de noter que les différences dans la formation des entérotoxines étaient davantage liées au type de souches qu'à la température. Ces résultats ont permis de conclure que le mécanisme de formation des entérotoxines dans le fromage devait être analysé de manière plus approfondie. Étant donné que le sujet est très vaste et que les connaissances sur l'analyse des entérotoxines staphylococciques sont très limitées, il a fallu trouver un partenaire de recherche afin de combler ces lacunes.

Le partenaire de recherche idéal a été trouvé avec Anses et le « Laboratoire de sécurité des aliments de Maisons-Alfort » qui fait partie de l'unité Staphylococcus, bacillus, clostridies (SBCL). Ce laboratoire de sécurité alimentaire contribue à améliorer la qualité des aliments et le contrôle de l'hygiène grâce à son expertise en matière de développement analytique. Il apporte un soutien aux décideurs publics. L'unité SBCL effectue des études sur les staphylocoques à coagulase positive (SCP), les bacilles, les clostridies impliqués dans les intoxications alimentaires. L'unité SBCL a été nommée laboratoire de référence européen EURL (European Union Reference Laboratories) pour les SCP. Elle gère les réseaux NRL (National Reference Laboratory), accomplit des activités de référence, y compris le développement et la validation de méthodes, et organise des essais d'aptitude.

La collaboration entre Agroscope et Anses a été le point de départ de cette thèse. Après une recherche bibliographique approfondie, les méthodes et les souches disponibles, l'objectif et la problématique de recherche spécifiques ont été définis. Tels étaient les contenus de la présente thèse de doctorat.

2 Theoretical background

Staphylococcus aureus

Characteristics and epidemiology of S. aureus

In 1881 Staphylococcus was discovered by Sir Alexander Ogston describing staphylococcal disease and its role in sepsis [START_REF] Ogston | Micrococcus Poisoning[END_REF]. In 1884, a German scientist (Friedrich Julius

Rosenbach) was able to distinguish Staphylococcus aureus from Staphylococcus albus colonies, because of their different colours, S. albus was later renamed S. epidermis [START_REF] Licitra | Etymologia:Staphylococcus[END_REF].

S. aureus is member of the Micrococcaceae family and on microscopical examination, the organisms appear as Gram-positive cocci in clusters. S. aureus can be distinguished from other staphylococcal species because of the gold pigmentation of colonies and positive results of coagulase, mannitol-fermentation, and deoxyribonuclease tests [START_REF] Somerville | The Biology of Staphylococci[END_REF].

S. aureus is considered as an ubiquitous opportunistic and very versatile pathogen [START_REF] Coelho | Short communication: profile of virulence factors of Staphylococcus aureus isolated from subclinical bovine mastitis in the state of Rio de Janeiro, Brazil[END_REF][START_REF] Feng | Evolution and pathogenesis of Staphylococcus aureus: lessons learned from genotyping and comparative genomics[END_REF]. The microorganism is able to survive in hostile environments, colonizing the skin and mucous membranes, and causing various infections in humans and animals [START_REF] Coelho | Short communication: profile of virulence factors of Staphylococcus aureus isolated from subclinical bovine mastitis in the state of Rio de Janeiro, Brazil[END_REF][START_REF] Malachowa | Mobile genetic elements of Staphylococcus aureus[END_REF]. S. aureus is facultative anaerobe, and it can grow in a wide range of environmental conditions (Table 1). Doubling time in optimal laboratory condition has been described between 24 and 30 min and in vivo 60 min [START_REF] Domingue | Bacterial doubling time modulates the effects of opsonisation and available iron upon interactions between Staphylococcus aureus and human neutrophils[END_REF]. Because of the adaptive capacity of Staph. aureus the species has demonstrated its success as a pathogen [START_REF] Liu | Enterotoxin-Producing Staphylococcus aureus[END_REF]. It can lead to suppurative skin and soft tissue infections, which can be superficial (e.g. impetigo, furuncles), deep (e.g. abscess, osteomyelitis), systemic (bacteraemia), or foreign-body-related (Foster and Geoghegan, 2015). Further diseases caused by S. aureus include respiratory diseases (e.g. pneumonia) and toxin-related diseases such as food poisoning, the toxic shock syndrome, and the scalded skin syndrome (Foster and Geoghegan, 2015).

Humans are a natural reservoir of S. aureus. 30 to 50 % of healthy adults are colonized with this microorganism [START_REF] Chen | Resistance of enterococci to antibiotic combinations[END_REF][START_REF] Noble | Carriage of Staphylococcus aureus in random samples of a normal population[END_REF]. Out of them 10-20 % are persistently colonized with methicillin-sensitive and methicillin-resistant isolate [START_REF] Chen | Resistance of enterococci to antibiotic combinations[END_REF][START_REF] Sanford | Efficient detection and long-term persistence of the carriage of methicillin-resistant Staphylococcus aureus[END_REF]. Persons colonized with S. aureus are at increased risk for subsequent infection, especially patients with diabetes, intravenous drug users and patients undergoing haemodialysis, surgical patients and patients with the acquired immunodeficiency have an increased risk of staphylococcal disease [START_REF] Wenzel | The significance of nasal carriage of Staphylococcus aureus and the incidence of postoperative wound infection[END_REF].

Persons that are colonized with S. aureus strains are at increased risk of becoming infected with these strains. Most cases of nosocomial infection are acquired through exposure to the hands of health care workers after they have been transiently colonized with staphylococci from their own reservoir or from contact with an infected patient. Outbreaks may also result from exposure to a single long-term carrier or environmental sources, but these modes of transmission are less common [START_REF] Casewell | The carrier state: methicillin-resistant Staphylococcus aureus[END_REF][START_REF] Lowy | Staphylococcus aureus infections[END_REF].

In dairy cows, S. aureus represents one of the most prevalent mastitis pathogens worldwide [START_REF] Artursson | Genotyping of Staphylococcus aureus in bovine mastitis and correlation to phenotypic characteristics[END_REF]. This microorganism is able to produce a variety of toxins and virulence factors, which contribute to the establishment of a new infection, including colonization of the mammary gland, bacterial invasion of host cells, evasion of the host immune responses, virulence, and pathogenicity [START_REF] Artursson | Genotyping of Staphylococcus aureus in bovine mastitis and correlation to phenotypic characteristics[END_REF][START_REF] Coelho | Short communication: profile of virulence factors of Staphylococcus aureus isolated from subclinical bovine mastitis in the state of Rio de Janeiro, Brazil[END_REF].

S. aureus genome sequencing

The first genomes of S. aureus Mu50 and N315 were the first to be sequenced by [START_REF] Kuroda | Whole genome sequencing of meticillin-resistant Staphylococcus aureus[END_REF]. Since then a lot of genomic analysis were published about the characterisation of this species [START_REF] Baba | Genome sequence of Staphylococcus aureus strain Newman and comparative analysis of staphylococcal genomes: polymorphism and evolution of two major pathogenicity islands[END_REF][START_REF] Baba | Genome and virulence determinants of high virulence community-acquired MRSA[END_REF][START_REF] Feng | Evolution and pathogenesis of Staphylococcus aureus: lessons learned from genotyping and comparative genomics[END_REF][START_REF] Malachowa | Mobile genetic elements of Staphylococcus aureus[END_REF] (Figure 1). The S. aureus genome is circular and consists of about 2.8 Mb and contains approximately 2,600 genes, and its structure is highly conserved with small variable regions (Foster and Geoghegan, 2015). Genes governing virulence and resistance to antibiotics are found on the chromosome, as well as the on extrachromosomal elements [START_REF] Novick | The staphylococcus as a molecular genetic system[END_REF]. These genes are transferred between staphylococcal strains, species, or other Gram-positive bacterial species through the extrachromosomal elements [START_REF] Schaberg | Intergeneric and interspecies gene exchange in grampositive cocci[END_REF]) (Figure 2). The core genome (genes present in all strains of a species) makes up about 75% of the total S. aureus genome, and besides housekeeping genes encoding for essential growth and survival functions, it also includes genes expressing virulence factors like surface proteins, toxins, and enzymes (Foster and Geoghegan, 2015;[START_REF] Malachowa | Mobile genetic elements of Staphylococcus aureus[END_REF]. Variation in the core genome mostly results from single nucleotide polymorphisms (SNPs) or from differences in the length of repetitive regions (Foster and Geoghegan, 2015). S. aureus is further characterized by the presence of mobile genetic elements (MGEs), which make up about 25% of the staphylococcal genome [START_REF] Malachowa | Mobile genetic elements of Staphylococcus aureus[END_REF].

The diversity of MGEs in S. aureus genomes

MGEs in S. aureus are putative and proven virulence factors with genes responsible for host adaptation and toxin production [START_REF] Baba | Genome sequence of Staphylococcus aureus strain Newman and comparative analysis of staphylococcal genomes: polymorphism and evolution of two major pathogenicity islands[END_REF][START_REF] Baba | Genome and virulence determinants of high virulence community-acquired MRSA[END_REF][START_REF] Lindsay | Staphylococcus aureus: superbug, super genome?[END_REF]. Following MGEs are present in S. aureus genome (Figure 3): Bacteriophages, Pathogenicity islands, Genomic islands, Plasmids, Transposon, insertation sequence and Staphylococcal cassette chromosome.

Bacteriophages and virulence

Bacteriophages (phages) or bacterial viruses seem to have an impact on staphylococcal diversity and evolution. Phages are classified into three distinctive groups: lytic, temperate, and chronic [START_REF] Malachowa | Mobile genetic elements of Staphylococcus aureus[END_REF]. Lytic phages are members of the Myoviridae family that has been used in phage therapy, because bacteria lyse completely during release of progeny phages. Bacteria infected with chronic phages release progeny into the extracellular environment without killing the host, which allows bacteria to grow and divide [START_REF] Malachowa | Mobile genetic elements of Staphylococcus aureus[END_REF]. Temperate phages, which are members of the Siphoviridae family, form the most numerous group among all S. aureus phages. Temperate phages have the ability to lyse bacteria after infection, but they typically form a long-term relationship with the host cell, whereby the phage DNA integrates into the staphylococcal genome as a prophage [START_REF] Goerke | Diversity of prophages in dominant Staphylococcus aureus clonal lineages[END_REF][START_REF] Mann | The potential of phages to prevent MRSA infections[END_REF]. Phage can impact expression of virulence determinants by positive or negative lysogenic conversion. With a positive conversion, bacteria express prophage-encoded virulence determinants, for negative conversion there is an insertional inactivation of genes [START_REF] Coleman | Staphylococcus aureus bacteriophages mediating the simultaneous lysogenic conversion of beta-lysin, staphylokinase and enterotoxin A: molecular mechanism of triple conversion[END_REF][START_REF] Goerke | Diversity of prophages in dominant Staphylococcus aureus clonal lineages[END_REF]. Prophageencoded virulence molecules in S. aureus are enterotoxins or Panton-Valentin leucocidin [START_REF] Malachowa | Mobile genetic elements of Staphylococcus aureus[END_REF]. Prophage can also work with other MGEs by promoting mobility of some staphylococcal pathogenicity islands or by transferring antibiotic resistance by transduction of plasmids previously incorporated into chromosomal DNA [START_REF] Fitzgerald | Characterization of a putative pathogenicity island from bovine Staphylococcus aureus encoding multiple superantigens[END_REF][START_REF] Novick | Mobile genetic elements and bacterial toxinoses: the superantigenencoding pathogenicity islands of Staphylococcus aureus[END_REF].

Pathogenicity islands

Staphylococcal pathogenicity islands (SaPIs) are MGEs of 14-17 kb in size [START_REF] Malachowa | Mobile genetic elements of Staphylococcus aureus[END_REF]. To date, at least 16 SaPIs have been sequenced and SaPI1 (Figure 5) is considered as the prototype [START_REF] Novick | Mobile genetic elements and bacterial toxinoses: the superantigenencoding pathogenicity islands of Staphylococcus aureus[END_REF][START_REF] Novick | The SaPIs: mobile pathogenicity islands of Staphylococcus[END_REF]. SaPIs form a coherent family with highly conserved core genes [START_REF] Novick | The SaPIs: mobile pathogenicity islands of Staphylococcus[END_REF][START_REF] Ubeda | Sip, an integrase protein with excision, circularization and integration activities, defines a new family of mobile Staphylococcus aureus pathogenicity islands[END_REF]. Core

genes include two open reading frames encoding transcriptional regulatory proteins and a region encoding integrase, Rep protein, and terminase. In addition to core genes, almost all SaPIs encode enterotoxins or toxic shock syndrome toxin (TSST) [START_REF] Yarwood | Characterization and expression analysis of Staphylococcus aureus pathogenicity island 3. Implications for the evolution of staphylococcal pathogenicity islands[END_REF].

SaPIbov2 is an exception to this rule, and instead it contains Bap adhesion protein, which plays a role in bovine chronic mastitis infections [START_REF] Tormo | Bap-dependent biofilm formation by pathogenic species of Staphylococcus: evidence of horizontal gene transfer?[END_REF][START_REF] Ubeda | Sip, an integrase protein with excision, circularization and integration activities, defines a new family of mobile Staphylococcus aureus pathogenicity islands[END_REF].

Genomic islands

Three classes of genomic islands exist among the S. aureus strains whose genomes have been sequenced [START_REF] Baba | Genome and virulence determinants of high virulence community-acquired MRSA[END_REF][START_REF] Diep | Complete genome sequence of USA300, an epidemic clone of community-acquired meticillin-resistant Staphylococcus aureus[END_REF][START_REF] Klaui | Characterization and Comparative Analysis of the Staphylococcus aureus Genomic Island vSabeta: an In Silico Approach[END_REF][START_REF] Lindsay | Staphylococcus aureus: superbug, super genome?[END_REF]. These genomic islands are named: vSaα, vSaβ, and vSaγ. These genomic islands are extremely stable and have highly conserved genes. The gene content is usually highly conserved but can also vary substantially between some strains. [START_REF] Baba | Genome sequence of Staphylococcus aureus strain Newman and comparative analysis of staphylococcal genomes: polymorphism and evolution of two major pathogenicity islands[END_REF][START_REF] Baba | Genome and virulence determinants of high virulence community-acquired MRSA[END_REF]. A current notion is that genomic islands were once mobile elements acquired by horizontal gene [START_REF] Waldron | Sau1: a novel lineage-specific type I restrictionmodification system that blocks horizontal gene transfer into Staphylococcus aureus and between S. aureus isolates of different lineages[END_REF], a new study suggest that vSaβ is a prophage that can still be mobilized [START_REF] Klaui | Characterization and Comparative Analysis of the Staphylococcus aureus Genomic Island vSabeta: an In Silico Approach[END_REF].

These genomic islands are flanked by a broken transposase gene upstream and partial restriction-modification system (RM) type I downstream. A complete RM type I comprises host specificity determinant genes hsdR, hsdM, and hsdS, but only hsdM and hsdS are found juxtaposed to the S. aureus genomic islands. Both flanking DNA segments contribute to the stability of genomic islands within the S. aureus chromosome [START_REF] Baba | Genome and virulence determinants of high virulence community-acquired MRSA[END_REF][START_REF] Ito | Insights on antibiotic resistance of Staphylococcus aureus from its whole genome: genomic island SCC[END_REF][START_REF] Waldron | Sau1: a novel lineage-specific type I restrictionmodification system that blocks horizontal gene transfer into Staphylococcus aureus and between S. aureus isolates of different lineages[END_REF].

About vSaα it is known that they harbour a lipoprotein gene cluster (lpl) and staphylococcal superantigen-like genes (ssl) [START_REF] Lina | Standard nomenclature for the superantigens expressed by Staphylococcus[END_REF] and about vSaγ it is known for containing genes encoding b-type phenol-soluble modulins and a cluster of ssl genes similar to that present within vSaα [START_REF] Gill | Insights on evolution of virulence and resistance from the complete genome analysis of an early methicillin-resistant Staphylococcus aureus strain and a biofilm-producing methicillin-resistant Staphylococcus epidermidis strain[END_REF].

The genomic island vSaβ (Figure 3) is of particular interest, as it carries two genes belonging to the type I staphylococcal restriction-modification system (hsdM and hsdS) and harbours a number of virulence-associated genes, such as a hyaluronate lyase precursor gene (hysA), a lantibiotic gene cluster (bacteriocins of S. aureus [bsa]), two leukocidin genes (lukD and lukE), an enterotoxin gene cluster (egc), and a cluster of serine protease genes (serine protease like

[spl] genes) [START_REF] Baba | Genome sequence of Staphylococcus aureus strain Newman and comparative analysis of staphylococcal genomes: polymorphism and evolution of two major pathogenicity islands[END_REF].

In older study, three different kinds of vSaβ were found [START_REF] Baba | Genome sequence of Staphylococcus aureus strain Newman and comparative analysis of staphylococcal genomes: polymorphism and evolution of two major pathogenicity islands[END_REF]. Nowadays with the new opportunities offered by whole genome sequencing a recent study [START_REF] Klaui | Characterization and Comparative Analysis of the Staphylococcus aureus Genomic Island vSabeta: an In Silico Approach[END_REF] identified 15 new types of vSaβ out of 103 clinical S. aureus strains (Figure 4). These new insights give a superordinate system to classify S. aureus strains based on genomic islands [START_REF] Klaui | Characterization and Comparative Analysis of the Staphylococcus aureus Genomic Island vSabeta: an In Silico Approach[END_REF]. plasmids, which usually carry several resistance determinants; and (3) conjugative multiresistance plasmids [START_REF] Berg | Complete nucleotide sequence of pSK41: evolution of staphylococcal conjugative multiresistance plasmids[END_REF]. Larger plasmids undergo theta replication (a DNA replication mechanism that resembles the Greek letter theta), whereas small plasmids usually replicate by the rolling-circle mechanism [START_REF] Khan | Plasmid rolling-circle replication: highlights of two decades of research[END_REF][START_REF] Lindsay | Genomic variation and evolution of Staphylococcus aureus[END_REF]. As a consequence of the limited ability of S. aureus to acquire DNA from the environment (low natural competence)

compared to bacteria such as Vibrio cholera or Bacillus subtilis, most of the intercellular transfer of staphylococcal plasmids occurs by transduction or conjugation [START_REF] Morikawa | A new staphylococcal sigma factor in the conserved gene cassette: functional significance and implication for the evolutionary processes[END_REF]. Upon entering the bacterial host, staphylococcal plasmids remain as free circularized DNA or linearize and integrate into the chromosome [START_REF] Malachowa | Mobile genetic elements of Staphylococcus aureus[END_REF].

Staphylococcal plasmids are known for their genes encoding antibiotic resistance as for example penicillin and more recently vancomycin, which resistance was acquired from enterococci [START_REF] Weigel | Genetic analysis of a high-level vancomycin-resistant isolate of Staphylococcus aureus[END_REF]Zhu et al., 2008).

In addition to genes encoding antibiotic resistance and molecules involved in metabolism, staphylococcal plasmids encode resistance to a variety of organic and inorganic ions, such as cadmium, mercury, arsenate, etc., which are highly toxic for living cells [START_REF] Jensen | Genetics of antimicrobial resistance in Staphylococcus aureus[END_REF]. Staphylococcal plasmids may also encode toxin genes like plasmids containing genes encoding enterotoxins. exfoliative toxin B, bacteriocin, and bacteriocin immunity [START_REF] Jackson | Cloning and expression of the exfoliative toxin B gene from Staphylococcus aureus[END_REF].

Staphylococcal cassette chromosome

Staphylococcal cassette chromosomes (SCCs) are relatively large fragments of DNA that always insert into the orfX gene on the S. aureus chromosome. SCC can encode antibiotic resistance and/or virulence determinants, SCCs can be classified into staphylococcal cassette chromosome mec (SCCmec) or non-SCCmec groups [START_REF] Bayles | Genetic and molecular analyses of the gene encoding staphylococcal enterotoxin D[END_REF][START_REF] Malachowa | Mobile genetic elements of Staphylococcus aureus[END_REF]. MRSA strains contain SCCmec encoding the methicillin resistance gene (mecA), thus conferring resistance to methicillin and to all β-lactam antibiotics [START_REF] Malachowa | Mobile genetic elements of Staphylococcus aureus[END_REF].

Staphylococcal cassette chromosomes can be complex and are thus not limited to encoding methicillin resistance. Non-mec SCC and wSCC (without or no functional recombinase) contain virulence or fitness/survival determinants [START_REF] Malachowa | Mobile genetic elements of Staphylococcus aureus[END_REF]. A non-mec SCC is the one containing the adlb gene, which is highly sensitive and specific for S. aureus genotype B (GTB, a genotype often involved in mastitis), both at the analytical and the diagnostic level, enabling the very specific detection of the genotype of interest [START_REF] Sartori | Development of a new real-time quantitative PCR assay for the detection of Staphylococcus aureus genotype B in cow milk, targeting the new gene adlb[END_REF].

Transposon and insertation sequence

Although insertion sequences (IS) can exist independently in the S. aureus genome, they often present as pairs constituting a composite transposon [START_REF] Byrne | Nucleotide sequence analysis of IS256 from the Staphylococcus aureus gentamicin-tobramycin-kanamycin-resistance transposon Tn4001[END_REF]. IS insert into various loci and may cause changes in the expression of genes in the core chromosome. Transposons and insertation sequences predominantly encode antibiotic resistance genes in S. aureus, but they can also encode for staphylococcal enterotoxin (seh) [START_REF] Ren | Characterization and biological properties of a new staphylococcal exotoxin[END_REF]. The smaller transposons are usually presented in multiple copies in the staphylococcal genome, either inserted into the chromosome or into MGEs, such as SCC or plasmids. This group includes Tn554 and Tn552, which encode resistance to MLSB antibiotics and spectinomycin or penicillinase, respectively [START_REF] Bagcigil | Occurrence, species distribution, antimicrobial resistance and clonality of methicillin-and erythromycin-resistant staphylococci in the nasal cavity of domestic animals[END_REF][START_REF] Ito | Insights on antibiotic resistance of Staphylococcus aureus from its whole genome: genomic island SCC[END_REF].

Acquisition of MGE by horizontal gene transfer (HGT) allows S. aureus strains to have advantages for survival at a wide range of environmental conditions, including interspecies competition within particular ecological niche and antibiotic selective pressure. Although MGEs constitute only 25% of the staphylococcal genome, they encode many putative virulence factors and antibiotic determinants and thus play an important role in bacterial adaptability and survival [START_REF] Lindsay | Staphylococcus aureus: superbug, super genome?[END_REF][START_REF] Malachowa | Mobile genetic elements of Staphylococcus aureus[END_REF].

Staphylococcal enterotoxins

Overview

S. aureus is a versatile pathogen that produces a wide range of exoproteins with toxicological effects on humans and animals, such as hyaluronidase, staphylokinase, nucleases, lipases, proteases, collagenases, hemolysins, exfoliative toxins, and superantigen proteins encompassing the Toxic Shock Syndrome Toxin-1 (TSST-1), Staphylococcal Enterotoxin-like proteins (SEls) and staphylococcal enterotoxins (SEs). Among these exotoxins, SEs have been the most frequently associated with food-borne intoxications collectively referred to as Staphylococcal Foodborne Poisoning (SFP) (Marie-Laure De [START_REF] Buyser | Implication of milk and milk products in food-borne diseases in France and in different industrialised countries[END_REF]. Staphylococcal enterotoxin was first described in 1959 [START_REF] Bergdoll | Staphylococcus aureus in[END_REF].

SEs and SEls are of low molecular weight, single-chain basic globular proteins that specifically attack intestinal cells leading to gastroenteritis, typically evoking vomiting, diarrhoea and intestinal or gastric inflammation. They have biochemical and structural properties that make them resistant to heat, digestive proteinases, irradiation, denaturing agents, and stable a wide pH range [START_REF] Asao | An extensive outbreak of staphylococcal food poisoning due to low-fat milk in Japan: estimation of enterotoxin A in the incriminated milk and powdered skim milk[END_REF][START_REF] Fung | Thermal inactivation of staphylococcal enterotoxin B and C[END_REF]Hennekinne et al., 2012).

Staphylococcal enterotoxin genes are also harbored by other Staphylococci, like:

Staphylococcus cohnii, Staphylococcus epidermidis, Staphylococcus xylosus and

Staphylococcus haemolyticus, Staphylococcus argentus and Staphylococcus schweitzeri,

Staphylococcus carnosus and Staphylococcus warneri [START_REF] Bautista | A quantitative study of enterotoxin production by sheep milk staphylococci[END_REF][START_REF] Chajecka-Wierzchowska | Enterotoxigenic Potential of Coagulase-Negative Staphylococci from Ready-to-Eat Food[END_REF][START_REF] Da Cunha Mde | Detection of enterotoxin and toxic shock syndrome toxin 1 genes in Staphylococcus, with emphasis on coagulase-negative staphylococci[END_REF][START_REF] Veras | A study of the enterotoxigenicity of coagulase-negative and coagulase-positive staphylococcal isolates from food poisoning outbreaks in Minas Gerais, Brazil[END_REF][START_REF] Zell | Characterization of toxin production of coagulase-negative staphylococci isolated from food and starter cultures[END_REF][START_REF] Zhang | Identification and characterization of two novel superantigens among Staphylococcus aureus complex[END_REF]. Recently for these coagulase negative staphylococci, their potential production of enterotoxin was studied and it was proven that they are not able to produce enterotoxins [START_REF] Chajecka-Wierzchowska | Enterotoxigenic Potential of Coagulase-Negative Staphylococci from Ready-to-Eat Food[END_REF]. Nevertheless the authors suggest that due to the high presence of staphylococcal enterotoxin genes coagulase in negative staphylococci should not be ignored because of their pathogenic potential [START_REF] Chajecka-Wierzchowska | Enterotoxigenic Potential of Coagulase-Negative Staphylococci from Ready-to-Eat Food[END_REF].

Characteristics of the different staphylococcal enterotoxins

By now 26 different types of Staphylococcal enterotoxin have been described and the number seems constantly on the rise, due to the wider use of automated characterisation techniques at the molecular and genetic levels (Table 2). Such sophisticated, rapid and sensible techniques are expected to allow identification of new SEs/SEls or reconsideration of the classification of some formerly classified SEs/SEls [START_REF] Benkerroum | Staphylococcal enterotoxins and enterotoxin-like toxins with special reference to dairy products: An overview[END_REF].

In 2004, the International Nomenclature Committee for Staphylococcal Superantigens (INCSS)

proposed a standard nomenclature for newly discovered toxins. The INCSS naming convention is to emphasize the relevance of the food poisoning (emetic activity). To name the SE, it is required to demonstrate emetic activity via the oral route in a primate model. If an SE exhibits no emetic potential in the vomiting model or its emetic activity is not yet examined, the toxin would be named staphylococcal like enterotoxins (SEl) [START_REF] Lina | Standard nomenclature for the superantigens expressed by Staphylococcus[END_REF]. In the past SEC was often divided into further subgroups like SEC1-3, SECbovine ,SECovine and SECcaprine [START_REF] Bohach | Nucleotide sequence of the staphylococcal enterotoxin C1 gene and relatedness to other pyrogenic toxins[END_REF][START_REF] Hovde | Investigation of the role of the disulphide bond in the activity and structure of staphylococcal enterotoxin C1[END_REF][START_REF] Marr | Characterization of novel type C staphylococcal enterotoxins: biological and evolutionary implications[END_REF]. Due to the high similarity at amino acid level, this subgrouping is not anymore made and the enterotoxin is considered as one single type (SEC) [START_REF] Liu | Enterotoxin-Producing Staphylococcus aureus[END_REF]. In 2020 a new enterotoxin was discovered named SE02 [START_REF] Suzuki | A novel staphylococcal enterotoxin SE02 involved in a staphylococcal food poisoning outbreak that occurred in Tokyo in 2004[END_REF]. By looking in detail at this enterotoxin it can be observed, that it has a great similarity at amino acids level to SEA. For this reason it was not taken into consideration in Table 2. As described in Table 2, enterotoxins can be part of different MGE, these are graphically summarized in Figure 5. A special case are also the enterotoxins located on the enterotoxin gene cluster (egc). These enterotoxins (seg, sei, sem, sen and seo) were first described in 2001 [START_REF] Jarraud | egc, a highly prevalent operon of enterotoxin gene, forms a putative nursery of superantigens in Staphylococcus aureus[END_REF].

Later on, on the same cluster also seu was described (C. Letertre, 2003). The egc is described to be located on the vSaβ, a genomic island of S. aureus (see 2.1.2) [START_REF] Baba | Genome sequence of Staphylococcus aureus strain Newman and comparative analysis of staphylococcal genomes: polymorphism and evolution of two major pathogenicity islands[END_REF].

According to literature, there are different variants of egc type and they have been named egc1 to egc4 (Figure 5) [START_REF] Collery | Associations between enterotoxin gene cluster types egc1, egc2 and egc3, agr types, enterotoxin and enterotoxin-like gene profiles, and molecular typing characteristics of human nasal carriage and animal isolates of Staphylococcus aureus[END_REF], recently also two more (egc5, egc6) were described [START_REF] Chieffi | Novel insights into the enterotoxigenic potential and genomic background of Staphylococcus aureus isolated from raw milk[END_REF].

The difference between the egc types is that one or more of the enterotoxin genes of the egc have some variants of it or are deleted. An example is selu: In the egc where selu is not present, two pseudogenes are present instead: ent1 and ent2 (egc1). In literature selu is described as enterotoxin gene that was formed by a fusion of ent1 and ent2 (egc2). Egc3 was described as cluster having variants of each enterotoxin present (including seu). The last variant described is egc4 were sem and seu are not present and instead the presence of new enterotoxins selu2 or selw and selv was described. For this last egc type selv was described to be a recombination of sem and sei and selu2 as a fusion of ent1 and ent2 [START_REF] Thomas | Staphylococcal enterotoxin-like toxins U2 and V, two new staphylococcal superantigens arising from recombination within the enterotoxin gene cluster[END_REF].

According to more recent literature, 15 different vSaβ types were discovered, by analyzing 103 clinical S. aureus strains [START_REF] Klaui | Characterization and Comparative Analysis of the Staphylococcus aureus Genomic Island vSabeta: an In Silico Approach[END_REF]. Looking at these vSaβ, egc1 is described as vSaβ type I, egc2 as vSaβ type IV, egc3 as vSaβ III and egc4 was not found in any of the analyzed genomes so no vSaβ type was given [START_REF] Klaui | Characterization and Comparative Analysis of the Staphylococcus aureus Genomic Island vSabeta: an In Silico Approach[END_REF]. According to literature there are much more variants of egc than egc1 to egc6, as there are vSaβ types containing single truncated or fragmented genes (vSaβ type IV, XI). In consequence it is questionable if egc 1-6 is the correct way to distinguish the different variants of the enterotoxin gene cluster. In consequence of the fact that seu2 (or selw) is a variant of seu, in Table 2 only seu was considered. Sev seem also not to be relevant, as it was not found in the strains analyzed by Kläui et al (2009) nor in the ones analyzed by [START_REF] Merda | NAuRA: Genomic Tool to Identify Staphylococcal Enterotoxins in Staphylococcus aureus Strains Responsible for FoodBorne Outbreaks[END_REF].

Molecular structure of staphylococcal enterotoxins

Staphylococcal enterotoxins are globular, single-chain proteins with molecular weights between 19 and 29 kDa and 168-261 amino acids long (Table 2). A significant degree of similarity occurs among the primary peptide sequences of enterotoxins. Overall, 15% of the residues are entirely conserved throughout the known SEs and SEls. Most of these residues are located either centrally or at the C terminus [START_REF] Dinges | Exotoxins of Staphylococcus aureus[END_REF]. SEs can be divided into four phylogenetic groups based on their primary amino acid sequences [START_REF] Thomas | Staphylococcal enterotoxin-like toxins U2 and V, two new staphylococcal superantigens arising from recombination within the enterotoxin gene cluster[END_REF] or a phylogenetic tree can be constructed based on amino acid sequences (Figure 6). The three-dimensional structures of SE are conform to common protein folding what has been confirmed using crystallographic studies by [START_REF] Swaminathan | Residues defining Vβ specificity in staphylococcal enterotoxins[END_REF]. The overall shape of SE molecules is ellipsoid, and they contain two unequal domains: A and B (Figure 7). The

Pathogenesis of staphylococcal enterotoxins

Staphylococcal enterotoxins have the ability to induce emetic activity beside superantigenic activity (Figure 8). These two activities are taking place in two separate domains of the protein [START_REF] Dinges | Exotoxins of Staphylococcus aureus[END_REF][START_REF] Hovde | Investigation of the role of the disulphide bond in the activity and structure of staphylococcal enterotoxin C1[END_REF]. The superantigenic activity has the ability to induce excessive activation of T-cells with a subsequent massive release of cytokine, whereas the emetic activity has the ability to cause enteritis in the intestine among other symptoms [START_REF] Benkerroum | Staphylococcal enterotoxins and enterotoxin-like toxins with special reference to dairy products: An overview[END_REF]. A summary of the tested superantigenic and emetic activity of the different staphylococcal enterotoxins can be seen in Table 3. Not for all known SEs emetic activity has been tested.

Superantigen activity

SEs and SEls are representative superantigenic toxins, which selectively activate a vast number of T cells, depending on Vβ elements in the β chain of a T-cell receptor (TCR), in direct association with the major histocompatibility complex (MHC) class II molecules on antigenpresenting cells (APCs) [START_REF] Lina | Standard nomenclature for the superantigens expressed by Staphylococcus[END_REF][START_REF] Uchiyama | Bacterial superantigens--mechanism of T cell activation by the superantigens and their role in the pathogenesis of infectious diseases[END_REF]. They subsequently stimulate massive cytokine release and systemic shock (Figure 9). SEs have evolved in several distinct modes of interaction with MHC class II molecules. SEA contains two MHC class II binding sites (Figure 9). The zinc-dependent site is the major interaction region, and several important residues (H187, H225, and D227) were identified by mutagenesis. It is presumed that this binds MHC β chain [START_REF] Schad | Crystal structure of the superantigen staphylococcal enterotoxin type A[END_REF]. This major MHC binding site is located in domain A, near the amino terminus [START_REF] Schad | Crystal structure of the superantigen staphylococcal enterotoxin type A[END_REF]. The second (minor) binding site on SEA is F47 located in domain B, which is not zinc dependent. It may be this cooperation between the two binding sites that is responsible for the high affinity of SEA for MHC class II molecules. It could result in a trimer containing the toxin and two bound MHC class II molecules. Mutational studies were done on SEC3 [START_REF] Leder | A mutational analysis of the binding of staphylococcal enterotoxins B and C3 to the T cell receptor beta chain and major histocompatibility complex class II[END_REF], and showed that mutations in the TCR binding site of this toxin were capable of sharply reducing mitogenicity.

Emetic activity

The first well-documented report that clearly identified SEs as the cause of food poisoning outbreaks, was done by [START_REF] Dack | An outbreak of food poisoning proved to be due to a yellow haemolytic Staphylococcus[END_REF]. They isolated a pigment-forming Staphylococcus present in large numbers in a Christmas cake responsible for a food poisoning incident, and sterile culture filtrate of the organism reproduced illness when ingested by human volunteers.

The target of SEs responsible for initiating the emetic reflex could be located in the abdominal viscera, where putative cellular receptors for SEs exist [START_REF] Sugiyama | Abdominal viscera as site of emetic action for staphylococcal enterotoxin in the monkey[END_REF]. Since these receptors have not yet been identified, there remains much uncertainty regarding the early events in the pathogenesis of food poisoning.

The symptoms of staphylococcal food poisoning are vomiting, abdominal cramps, nausea, sometimes followed by diarrhea after a short period of incubation [START_REF] Hu | Induction of emetic response to staphylococcal enterotoxins in the house musk shrew (Suncus murinus)[END_REF]Le Loir et al., 2003). In contrast to these well-described clinical manifestations, the physiopathology of symptoms is only partially understood However, little is known regarding the mechanisms by which they induce vomiting. Lack of progress in elucidating the mechanism of the emetic activity of SEs can be partially attributed to the lack of convenient and appropriate animal models. The susceptible animal species to develop human-like enterotoxigenic disease are non-human primate models, often, Macaca mulatta [START_REF] Normann | Pathology of experimental enterotoxemia. The in vivo localization of staphylococcal enterotoxin B[END_REF][START_REF] Stiles | Response of the rhesus monkey, Macaca mulatta, to continuously infused staphylococcal enterotoxin B[END_REF]. When introduced intragastrically, SEA and SEB have been shown to induce emetic responses and gastrointestinal (GI) inflammatory changes in different Macaca spp. [START_REF] Merrill | The effect of staphylococcal enterotoxin on the fine structure of the monkey jejunum[END_REF][START_REF] Reck | Protection against the staphylococcal enterotoxin-induced intestinal disorder in the monkey by anti-idiotypic antibodies[END_REF]. Monkeys have been considered to be the primary animal model. However, the use of monkeys in investigating SEs is severely restricted by the high cost, the availability of these animals, and ethical considerations.

The house musk shrew, Suncus murinus, has been described as a small animal model for the study of emetic response to various emetic drugs [START_REF] Chen | Ethanol-induced emesis in the house musk shrew, Suncus murinus[END_REF][START_REF] Okada | Antiemetic effects of serotonergic 5-HT1Areceptor agonists in Suncus murinus[END_REF]. The emetic response of house musk shrew to peroral and intraperitoneal administration of SEA has been examined [START_REF] Hu | Analysis of the epitopes on staphylococcal enterotoxin A responsible for emetic activity[END_REF][START_REF] Hu | Emesis in the shrew mouse (Suncus murinus) induced by peroral and intraperitoneal administration of staphylococcal enterotoxin A[END_REF]) (Figure 10) The emetic activity of SEA in house musk shrews was found to be dose-dependent. Multiple emetic episodes occurred during 70 to 108 min after peroral administration of SEA. Similar responses occurred during 65 to 102 min after intraperitoneal injection of SEA [START_REF] Hu | Emesis in the shrew mouse (Suncus murinus) induced by peroral and intraperitoneal administration of staphylococcal enterotoxin A[END_REF]. The animals recovered clinically within 3 h. SEA passes through the mucosal epithelium in the GI lumen by an unknown mechanism and then accumulates in the submucosa (Figure 10). This translocation from the lumen to the submucosa occurs within 30-90 min, a timeframe that is consistent with the latency time of SEA-induced emesis in house musk shrew (30-120 min) [START_REF] Hu | Induction of emetic response to staphylococcal enterotoxins in the house musk shrew (Suncus murinus)[END_REF][START_REF] Hu | Emesis in the shrew mouse (Suncus murinus) induced by peroral and intraperitoneal administration of staphylococcal enterotoxin A[END_REF]. In the stomach and duodenum of house musk shrew, SEA binds to the submucosal mast cells or directly to neuron cells. The binding of SEA to an unidentified receptor expressed on the surface of these cells induces the degranulation, resulting in the release of 5-HT. At present, it is unclear what type of molecule acts as an SEA receptor on the surface of submucosal mast cells or neuron cells. Superantigens including SEA bind to MHC class II molecules expressed on surface of APCs. However, SEA and MHC class II signals were not co-localized in the GI tissues of the SEA administered animals, indicating that a receptor on mast cells is not MHC class II. Moreover, orally administrated SEA shows tendency to bind to mast cells rather than MHC class II-positive cells in GI tract, indicating that unidentified SEA receptor on mast cells is capable of binding SEA more efficiently than MHC class II [START_REF] Ono | Submucosal mast cells in the gastrointestinal tract are a target of staphylococcal enterotoxin type A[END_REF]. Further studies on the identification and molecular cloning of the unidentified SEA receptor gene are necessary for understanding the exact molecular basis of SEA-induced emesis and elucidation of its downstream intracellular signaling.

Prevalence of the different enterotoxins in S. aureus strains

Most of S. aureus genome contains one or more enterotoxin genes [START_REF] Merda | NAuRA: Genomic Tool to Identify Staphylococcal Enterotoxins in Staphylococcus aureus Strains Responsible for FoodBorne Outbreaks[END_REF]. Some of the genes seem to be more prevalent than the others and this seems to depend on the origin where the strains has been isolated [START_REF] Benkerroum | Staphylococcal enterotoxins and enterotoxin-like toxins with special reference to dairy products: An overview[END_REF]. A literature summary of the prevalence of the different genes harboured by different S. aureus strains can be seen in Table 4. Egc seems to be especially present in association with strains isolated from humans [START_REF] Jarraud | egc, a highly prevalent operon of enterotoxin gene, forms a putative nursery of superantigens in Staphylococcus aureus[END_REF]. In this study (from France) egc enterotoxins were found in 57% of the strains isolated from nasal carriage and in 67% of the strains from suppurative disease. In strains isolated from animals (from Ireland) egc enterotoxins were present highest (86.7%) on strains isolated from chicken and lowest (10.3%) in strains isolated from goats [START_REF] Smyth | Superantigen genes encoded by the egc cluster and SaPIbov are predominant among Staphylococcus aureus isolates from cows, goats, sheep, rabbits and poultry[END_REF].

From the non-classical enterotoxins, seiv wasn't found in none of the isolates analyzed.

Food borne outbreaks caused by Staphylococcal enterotoxins

The origins of staphylococcal food poisoning differ widely among countries what may be due to differences in consumption and food habits in each country. The main source of contamination are humans through contaminated food handlers via manual contact or via respiratory tract by coughing and sneezing. Often contamination occurs after heat treatment of the food. Contaminated food from animal origins, such as raw meat, sausages, raw milk, and raw milk cheese is frequently contaminated by animal carriage or infection by S. aureus like mastitis (Le [START_REF] Loir | Staphylococcus and food poisoning[END_REF] (Figure 12). After contamination (animal or human), food is processed (e.g. heating, cooling) what can inhibit or enhance Staphylococcal enterotoxin production. Even if heating is applied after growing of S. aureus, enterotoxins will still remain in the food, as the enterotoxins are heat stable. The intake of SE will then be the cause of Staphylococcal food poisoning [START_REF] Hennekinne | Staphylococcus aureus as a Leading Cause of Foodborne Outbreaks Worldwide, Staphylococcus aureus[END_REF].

Summarizing following series of events need to take place in order to qualify for an SFPO: (i)

presence of the pathogen in raw materials or the food handler(s), (ii) contamination of the food, for example, through processing equipment or through the food preparer, (iii) inappropriate storage conditions and/or inadequate temperature control that allows for bacterial growth and enterotoxin production, and (iv) ingestion of contaminated food containing a sufficient amount of SE to trigger symptoms of the disease [START_REF] Zeaki | The Role of Regulatory Mechanisms and Environmental Parameters in Staphylococcal Food Poisoning and Resulting Challenges to Risk Assessment[END_REF].

Overview of SFPO: EFSA Report

Outbreaks originating from bacterial toxin (other than Clostridium botulinum) in Europe seem to have slightly increased between 2017 and 2018 (Figure 13). Bacterial toxins considered in this case are toxins from Bacillus, Staphylococcus and Clostridium (other than C. botulinum). In 2018 114 outbreaks caused by Staphylococcal enterotoxin were reported, Out of them 37

were strong-evidence outbreaks and 77 weak evidence outbreaks. These outbreaks caused 1'124 human cases, out of them 167 people needed hospitalisation. The average number of people per outbreak is 9.9. In addition there are 636 weak evidence outbreaks caused by an unspecified bacterial toxin. Most of the outbreaks are reported in France due to the fact that in

France SFP is a notifiable disease. South European countries in general report more SFPs than Nordic countries, probably due to inappropriate storage conditions under more critical climate conditions.

Looking in detail which kind of foods are involved in the foodborne outbreaks (Figure 14) it seems that mixed food is the most involved one, followed by "other food, meat/meat products, and milk/milk products. Buffets meals, eggs/eggs products and fish/fisheries seem to be less involved [START_REF] Food Safety | The European Union One Health 2018 Zoonoses Report[END_REF]. In addition, it is remarkable that outbreaks caused by Staphylococcal enterotoxin and mixed food/meat products belong to the top-10 pathogen/food vehicle pair causing the highest number of hospitalisations. According to EFSA this number is increasing if compared to the numbers of 2010-2017. The category "mixed food" and "other foods" are both a miscellaneous group of foodstuffs including a large variety of multi-ingredients and multiorigin items. This heterogeneity makes it difficult to identify and trace back the primary food source contributing to the contamination. Mixed food was in 2018 also responsible for the highest number of illnesses and deaths among strong-evidence outbreaks at the EU level [START_REF] Food Safety | The European Union One Health 2018 Zoonoses Report[END_REF].

Involvement of the different enterotoxins in foodborne outbreaks

In literature outbreaks involving classical enterotoxins (SEA-SEE) are largely described.

Beside the ones described in Table 5 also older literature are describing outbreaks caused by SEA-SEE [START_REF] Kerouanton | Characterization of Staphylococcus aureus strains associated with food poisoning outbreaks in France[END_REF]. Especially SEA seems to have the ability to cause very large outbreak, causing more than 10'000 cases [START_REF] Asao | An extensive outbreak of staphylococcal food poisoning due to low-fat milk in Japan: estimation of enterotoxin A in the incriminated milk and powdered skim milk[END_REF][START_REF] Ikeda | Mass outbreak of food poisoning disease caused by small amounts of staphylococcal enterotoxins A and H[END_REF].

SEA-SEE are enterotoxins that can be analysed with commercially available methods like VIDAS SET2 or Ridascreen that is why this enterotoxin can easily be discovered and described [START_REF] Benkerroum | Staphylococcal enterotoxins and enterotoxin-like toxins with special reference to dairy products: An overview[END_REF]. Even classical enterotoxin can be detected and their respective enterotoxins gene detected too, often the strains involved are also harbouring non-classical enterotoxin genes (Table 5). Because of the fact that the detection methods (such as ELISA or other methods) for non-classical enterotoxins are not commercially available (Zhao et al., 2017a;Zhao et al., 2017b), detection of these enterotoxins remains difficult and their involvement in foodborne outbreaks cannot fully be described and confirmed.

During the last years multiple cases where described where the involvement of non-classical enterotoxin seems to be described. Among the non-classical enterotoxins SEH and the EGC enterotoxins seem to be the most involved enterotoxins (Table 5). About SEH, two outbreaks were described where the only detected CPS was harbouring seh gene, in consequence only this enterotoxin could have been involved in the outbreak [START_REF] Ciupescu | Characterization of Staphylococcus aureus strains and evidence for the involvement of non-classical enterotoxin genes in food poisoning outbreaks[END_REF][START_REF] Jorgensen | An outbreak of staphylococcal food poisoning caused by enterotoxin H in mashed potato made with raw milk[END_REF]. In one outbreak (Mashed potato, Norway), a method was available for SEH detection and it could be detected [START_REF] Jorgensen | An outbreak of staphylococcal food poisoning caused by enterotoxin H in mashed potato made with raw milk[END_REF]. For EGC enterotoxins in multiple outbreaks there is evidence of their involvement. Here again, in the two studies found (totally 3 outbreaks), CPS involved were only harbouring egc genes, but due to the lack of appropriate methods for enterotoxin detection, they were never analysed [START_REF] Johler | Further evidence for staphylococcal food poisoning outbreaks caused by egc-encoded enterotoxins[END_REF][START_REF] Umeda | Molecular and epidemiological characterization of staphylococcal foodborne outbreak of Staphylococcus aureus harboring seg, sei, sem, sen, seo, and selu genes without production of classical enterotoxins[END_REF].

Regulation of enterotoxin expression

As described in previous chapters S. aureus strains may harbour one or more se/sel genes.

The harbouring of these genes does not automatically mean that they are able to express them, even under optimal conditions of growth [START_REF] Carfora | Enterotoxin genes, enterotoxin production, and methicillin resistance in Staphylococcus aureus isolated from milk and dairy products in Central Italy[END_REF]Omoe, Hu, et al., 2005;[START_REF] Omoe | Detection of seg, seh, and sei genes in Staphylococcus aureus isolates and determination of the enterotoxin productivities of S. aureus isolates Harboring seg, seh, or sei genes[END_REF]. This may be due to the complexity of the genetic expression of se/sel genes involving an intricate network of regulatory systems acting independently or in coordination [START_REF] Bronner | Regulation of virulence determinants in Staphylococcus aureus: complexity and applications[END_REF].

S. aureus has a complex network of regulatory pathways to control toxin production. In consequence it responds to changes in the environment using a combination of quorumsensing (QS) [START_REF] Waters | Quorum sensing: cell-to-cell communication in bacteria[END_REF] and other two-component systems (TCS), of which at least 16 have been discovered in S. aureus to date [START_REF] Haag | The Role of Two-Component Signal Transduction Systems in Staphylococcus aureus Virulence Regulation[END_REF], as well as many trans-acting regulatory proteins [START_REF] Bronner | Regulation of virulence determinants in Staphylococcus aureus: complexity and applications[END_REF]. S. aureus relies on these systems to quickly make changes in the regulation of genes associated with important physiological features, including drug resistance, metabolism, immune evasion, and virulence. Each system can directly or indirectly control the transcription of specific sets of genes. However, the regulation of one gene may be influenced by multiple systems, leading to additional layers of regulation [START_REF] Fisher | Basis of Virulence in Enterotoxin-Mediated Staphylococcal Food Poisoning[END_REF] .

The mechanisms controlling SE production in S. aureus are multiple and include se gene promoter regions, multiple global regulators of virulence, such as the accessory gene regulator (agr), the staphylococcal accessory regulator (sar), the repressor of toxins (rot), the σB factor, and the two component system S. aureus exoprotein expression (sae) (Figure 15).

These regulatory mechanisms are affected in different ways by the environmental conditions in a food product, like salt content, water activity, and pH [START_REF] Schelin | Expression of Staphylococcal Enterotoxins under Stress Encountered during Food Production and Preservation[END_REF]. This can have a further impact on the production of the respective enterotoxins, making them difficult to control [START_REF] Zeaki | The Role of Regulatory Mechanisms and Environmental Parameters in Staphylococcal Food Poisoning and Resulting Challenges to Risk Assessment[END_REF].

In addition, according to the actual knowledge, different mobile genetic elements seem to have different regulatory systems. These can be observed for SEA (prophage encoded) and SED (plasmid encoded) where the first one's regulation is linked to the life cycle of the bacteriophages and is partly regulated by the agr system [START_REF] Zeaki | The Role of Regulatory Mechanisms and Environmental Parameters in Staphylococcal Food Poisoning and Resulting Challenges to Risk Assessment[END_REF]. The consequences on enterotoxin production are that SEA is produced mostly during the exponential growth phase, at the peak of replication [START_REF] Derzelle | Differential temporal expression of the staphylococcal enterotoxins genes during cell growth[END_REF][START_REF] Schelin | The formation of Staphylococcus aureus enterotoxin in food environments and advances in risk assessment[END_REF], whereas more SED is produced during the transition from the exponential to the stationary growth phase of the microorganism [START_REF] Schelin | The formation of Staphylococcus aureus enterotoxin in food environments and advances in risk assessment[END_REF].

Description of the different regulation systems

The accessory gene regulator (Agr) is a quorum sensing system which is activated at high cell densities and is made of two transcriptional units transcribing in opposing directions: RNAII, which codes for four genes (agrA, agrB, agrC, and agrD) [START_REF] Novick | The agr P2 operon: an autocatalytic sensory transduction system in Staphylococcus aureus[END_REF] and RNAIII, a regulatory RNA. These transcripts are controlled by the promoters P2 and P3, respectively.

AgrD, which contains the sequence for the autoinducing peptide (AIP), is processed and exported out of the cell by the combined actions of the membrane-associated export protein, AgrB [START_REF] Ji | Bacterial interference caused by autoinducing peptide variants[END_REF][START_REF] Ji | Cell density control of staphylococcal virulence mediated by an octapeptide pheromone[END_REF] and a type I signal peptidase, SpsB [START_REF] Kavanaugh | A role for type I signal peptidase in Staphylococcus aureus quorum sensing[END_REF]. AIP acts as the ligand for the membrane bound histidine kinase, AgrC, leading to the phosphorylation of AgrA [START_REF] Ji | Cell density control of staphylococcal virulence mediated by an octapeptide pheromone[END_REF]. Activated AgrA binds to the P2 and P3 promoters, resulting in the perpetuation of a positive feedback loop [START_REF] Koenig | Staphylococcus aureus AgrA binding to the RNAIII-agr regulatory region[END_REF].

Expression of agr is affected by various trans-activating regulators, such as Sar family of regulatory proteins, (SarR, SarS, SarT, SarU, SarX, SarZ, SarV, MgrA, and Rot) [START_REF] Cheung | SarA of Staphylococcus aureus binds to the sarA promoter to regulate gene expression[END_REF][START_REF] Cheung | Cloning and sequencing of sarA of Staphylococcus aureus, a gene required for the expression of agr[END_REF][START_REF] Heinrichs | Characterization of the sar locus and its interaction with agr in Staphylococcus aureus[END_REF], σB [START_REF] Lauderdale | Interconnections between Sigma B, agr, and proteolytic activity in Staphylococcus aureus biofilm maturation[END_REF] and SrrAB (Staphylococcal respiratory response AB) [START_REF] Pragman | Characterization of virulence factor regulation by SrrAB, a two-component system in Staphylococcus aureus[END_REF][START_REF] Yarwood | Characterization and expression analysis of Staphylococcus aureus pathogenicity island 3. Implications for the evolution of staphylococcal pathogenicity islands[END_REF].

Additionally, σB and Rot can affect another important two component systems called SaeRS [START_REF] Kusch | The influence of SaeRS and sigma(B) on the expression of superantigens in different Staphylococcus aureus isolates[END_REF].

About σB is known to respond to high temperature, catabolites, alkaline pH, high salinity [START_REF] Kullik | The alternative sigma factor sigmaB in Staphylococcus aureus: regulation of the sigB operon in response to growth phase and heat shock[END_REF][START_REF] Pane-Farre | The sigmaB regulon in Staphylococcus aureus and its regulation[END_REF][START_REF] Wu | Sigma-B, a putative operon encoding alternate sigma factor of Staphylococcus aureus RNA polymerase: molecular cloning and DNA sequencing[END_REF] on the other side SarA largely responds to changes in microenvironments [START_REF] Cheung | Regulation of virulence determinants in vitro and in vivo inStaphylococcus aureus[END_REF]. SrrAB system has been shown to be particularly crucial for bacterial growth under anaerobic and hypoxic conditions [START_REF] Kinkel | The Staphylococcus aureus SrrAB two-component system promotes resistance to nitrosative stress and hypoxia[END_REF][START_REF] Mashruwala | The Staphylococcus aureus SrrAB Regulatory System Modulates Hydrogen Peroxide Resistance Factors, Which Imparts Protection to Aconitase during Aerobic Growth[END_REF][START_REF] Pragman | Characterization of virulence factor regulation by SrrAB, a two-component system in Staphylococcus aureus[END_REF][START_REF] Yarwood | Characterization and expression analysis of Staphylococcus aureus pathogenicity island 3. Implications for the evolution of staphylococcal pathogenicity islands[END_REF].

SaeRS (S. aureus exoprotein expression) system responds to membrane attack by antimicrobial molecules produced by the innate host defence [START_REF] Cho | Calprotectin Increases the Activity of the SaeRS Two Component System and Murine Mortality during Staphylococcus aureus Infections[END_REF][START_REF] Geiger | The virulence regulator Sae of Staphylococcus aureus: promoter activities and response to phagocytosis-related signals[END_REF][START_REF] Kuroda | Subinhibitory concentrations of betalactam induce haemolytic activity in Staphylococcus aureus through the SaeRS twocomponent system[END_REF][START_REF] Novick | Mobile genetic elements and bacterial toxinoses: the superantigenencoding pathogenicity islands of Staphylococcus aureus[END_REF]. Finally Rot, the global gene regulator [START_REF] Said-Salim | Global regulation of Staphylococcus aureus genes by Rot[END_REF] is known to be negatively regulated by RNAIII through an antisense mechanism [START_REF] Boisset | Staphylococcus aureus RNAIII coordinately represses the synthesis of virulence factors and the transcription regulator Rot by an antisense mechanism[END_REF][START_REF] Geisinger | Inhibition of rot translation by RNAIII, a key feature of agr function[END_REF].

Regulation of the different enterotoxins

This subchapter summarizes the actual state of the art literature. However, not for all enterotoxins information on the regulation mechanisms were available.

SEA

SEA is produced the highest during the exponential growth phase, at the peak of replication [START_REF] Derzelle | Differential temporal expression of the staphylococcal enterotoxins genes during cell growth[END_REF][START_REF] Schelin | The formation of Staphylococcus aureus enterotoxin in food environments and advances in risk assessment[END_REF]. The production of SEA was discovered to be closely tied to the phage's life cycle [START_REF] Cao | Elevated enterotoxin A expression and formation in Staphylococcus aureus and its association with prophage induction[END_REF] and to be inducible by bacterial stress [START_REF] Zeaki | Evaluation of Potential Effects of NaCl and Sorbic Acid on Staphylococcal Enterotoxin A Formation[END_REF].

Sea gene encoding for SEA is located on the genome of a polymorphic family of lysogenic bacteriophages, the Siphoviridae family. It has been proven, through a number of studies, that SEA-producing S. aureus strains can be categorized into high and low SEA producers, depending on the sea-carrying prophage they harbour [START_REF] Borst | Phage-associated differences in staphylococcal enterotoxin A gene (sea) expression correlate with sea allele class[END_REF][START_REF] Cao | Elevated enterotoxin A expression and formation in Staphylococcus aureus and its association with prophage induction[END_REF][START_REF] Wallin-Carlquist | Acetic acid increases the phage-encoded enterotoxin A expression in Staphylococcus aureus[END_REF]. [START_REF] Wallin-Carlquist | Acetic acid increases the phage-encoded enterotoxin A expression in Staphylococcus aureus[END_REF] showed that there are two sea variants: sea1 and sea2; the high-SEA-producing strains carried sea1, while sea2 was found in the low-SEA-producing strains (Figure 16). The state of lysogeny, although very stable, can be disrupted by certain environmental conditions (i.e., the presence of weak acids, high NaCl concentration, UV irradiation, and DNA damage by chemical agents), and in such cases, the lytic response is initiated by the phage; a process known as prophage induction [START_REF] Zeaki | The Role of Regulatory Mechanisms and Environmental Parameters in Staphylococcal Food Poisoning and Resulting Challenges to Risk Assessment[END_REF]. Prophage induction has been found to increase the amount of SEA produced by some SEA-producing strains, and thus increases the probability of SFPO [START_REF] Zeaki | The Role of Regulatory Mechanisms and Environmental Parameters in Staphylococcal Food Poisoning and Resulting Challenges to Risk Assessment[END_REF].

SEB, SEC, SED, SEJ

Early observations are showing that the production of SEB [START_REF] Czop | Staphylococcal enterotoxin synthesis during the exponential, transitional, and stationary growth phases[END_REF][START_REF] Derzelle | Differential temporal expression of the staphylococcal enterotoxins genes during cell growth[END_REF][START_REF] Gaskill | Regulation of the enterotoxin B gene in Staphylococcus aureus[END_REF], SEC [START_REF] Otero | Production of staphylococcal enterotoxins C1 and C2 and thermonuclease throughout the growth cycle[END_REF][START_REF] Regassa | Steady-state staphylococcal enterotoxin type C mRNA is affected by a product of the accessory gene regulator (agr) and by glucose[END_REF], and SED [START_REF] Bayles | Genetic and molecular analyses of the gene encoding staphylococcal enterotoxin D[END_REF] occurred between the exponential and the stationary phases of bacterial growth [START_REF] Gaskill | Regulation of the enterotoxin B gene in Staphylococcus aureus[END_REF][START_REF] Regassa | Steady-state staphylococcal enterotoxin type C mRNA is affected by a product of the accessory gene regulator (agr) and by glucose[END_REF][START_REF] Zhang | The enterotoxin D plasmid of Staphylococcus aureus encodes a second enterotoxin determinant (sej)[END_REF] suggesting that they could be regulated by Agr.

However, it was later shown that SEB, SEC and SED are regulated indirectly by additional factors. A summary of the factors involved in expression of these enterotoxins is illustrated in Table 6. [START_REF] Compagnone-Post | Role of host factors in the regulation of the enterotoxin B gene[END_REF][START_REF] Kusch | The influence of SaeRS and sigma(B) on the expression of superantigens in different Staphylococcus aureus isolates[END_REF][START_REF] Regassa | Steady-state staphylococcal enterotoxin type C mRNA is affected by a product of the accessory gene regulator (agr) and by glucose[END_REF][START_REF] Schmidt | Influences of sigmaB and agr on expression of staphylococcal enterotoxin B (seb) in Staphylococcus aureus[END_REF][START_REF] Tseng | Rot repression of enterotoxin B expression in Staphylococcus aureus[END_REF] SEC + + + nd nd [START_REF] Chien | SarA, a global regulator of virulence determinants in Staphylococcus aureus, binds to a conserved motif essential for sardependent gene regulation[END_REF][START_REF] Regassa | Steady-state staphylococcal enterotoxin type C mRNA is affected by a product of the accessory gene regulator (agr) and by glucose[END_REF][START_REF] Voyich | The SaeR/S gene regulatory system is essential for innate immune evasion by Staphylococcus aureus[END_REF] SED +/0 + --nd [START_REF] Sihto | Growth behavior and temporal enterotoxin D expression of Staphylococcus aureus strains under glucose and lactic acid stress[END_REF][START_REF] Sihto | Temporal expression of the staphylococcal enterotoxin D gene under NaCl stress conditions[END_REF][START_REF] Tseng | Accessory gene regulator control of staphyloccoccal enterotoxin d gene expression[END_REF] SEH SEH seems to be produced predominantly in the late exponential phase of bacterial growth and it seems to be agr independent [START_REF] Lis | Temporal expression of staphylococcal enterotoxin h in comparison with accessory gene regulator-dependent and -independent enterotoxins[END_REF][START_REF] Sakai | Characteristics of enterotoxin H-producing Staphylococcus aureus isolated from clinical cases and properties of the enterotoxin productivity[END_REF]. SEH was recently

shown to be positively regulated by Rot, via direct binding to the seh promoter [START_REF] Sato'o | Positive Regulation of Staphylococcal Enterotoxin H by Rot (Repressor of Toxin) Protein and Its Importance in Clonal Complex 81 Subtype 1 Lineage-Related Food Poisoning[END_REF], σB [START_REF] Kusch | The influence of SaeRS and sigma(B) on the expression of superantigens in different Staphylococcus aureus isolates[END_REF], several Sar homologs, and SaeR [START_REF] Kusch | The influence of SaeRS and sigma(B) on the expression of superantigens in different Staphylococcus aureus isolates[END_REF][START_REF] Sato'o | Positive Regulation of Staphylococcal Enterotoxin H by Rot (Repressor of Toxin) Protein and Its Importance in Clonal Complex 81 Subtype 1 Lineage-Related Food Poisoning[END_REF].

SElX

SaeRS appears to have a positive impact on SEX expression [START_REF] Langley | Staphylococcal enterotoxin-like X (SElX) is a unique superantigen with functional features of two major families of staphylococcal virulence factors[END_REF].

SEQ, SEK

SEK and SEQ, which are also found on sea-associated phages, can be transcriptionally induced by mitomycin C (Sumby and Waldor, 2003b).

EGC enterotoxins

The production of enterotoxins encoded in the egc operon (SEG, SEI, SEM, SEN, SEO, and SElU) is highest in the earliest stages of exponential growth [START_REF] Derzelle | Differential temporal expression of the staphylococcal enterotoxins genes during cell growth[END_REF] and dependent on σB [START_REF] Kusch | The influence of SaeRS and sigma(B) on the expression of superantigens in different Staphylococcus aureus isolates[END_REF].

Parameters influencing the production

Microorganisms in foods are affected by different parameters described as intrinsic factors (i.e., factors related to the food itself), extrinsic factors (i.e., factors related to the environment in which the food is stored), implicit factors (i.e., factors related to the microorganisms themselves), and finally processing factors (i.e., factors affecting the composition of the food and also the types and numbers of microorganisms that remain in the food after treatment) [START_REF] Hamad | Factors Affecting the Growth of Microorganisms in Food[END_REF]. Food matrixes are a complex system where the mentioned factors interact to a great extent. The data available at present are results from laboratory experiments, where the conditions were ideal [START_REF] Hennekinne | Staphylococcus aureus as a Leading Cause of Foodborne Outbreaks Worldwide, Staphylococcus aureus[END_REF]. A summary of factor affecting enterotoxin production of S. aureus is presented in Table 7.

Water activity (aW)

S. aureus can grow at a wide aw range, wider than most of the food-associated pathogens. S.

aureus can grow at a minimum aW of 0.83 (equivalent to about 20% NaCl) and has an optimum at an aw of >0.99. The conditions for SE production are slightly different that the ones of growing and depend on the type of SE [START_REF] Hennekinne | Staphylococcus aureus as a Leading Cause of Foodborne Outbreaks Worldwide, Staphylococcus aureus[END_REF].

SEA and SED production occurs under nearly all aw conditions allowing growth of S. aureus if all other conditions are optimal. Production of SEB is overly sensitive to reductions in aw and hardly any is produced at aw ≤0.93 despite extensive growth. The effect of aw on SEC production follows the same pattern as on SEB production [START_REF] Ewald | Effect of water activity on growth and enterotoxin D production of Staphylococcus aureus[END_REF][START_REF] Qi | Effect of low water activity on staphylococcal enterotoxin A and B biosynthesis[END_REF]. On the other side, SEE was found in media containing 10% NaCl (this concentration corresponds to aw 0.92) [START_REF] Thota | Effects of temperature, pH and NaCl on production of staphylococcal enterotoxins E and F[END_REF][START_REF] Troller | INFLUENCE OF WATER ACTIVITY ON GROWTH AND ENTEROTOXIN FORMATION BY Staphylococcus aureus IN FOODS[END_REF].

pH

Most staphylococcal strains grow at pH values between 4 and 10, with the optimum being 6-7. When other cultural parameters become nonoptimal, the pH range tolerated is reduced [START_REF] Hennekinne | Staphylococcus aureus as a Leading Cause of Foodborne Outbreaks Worldwide, Staphylococcus aureus[END_REF]. For example, the lowest pH that permitted growth and SE production by aerobically cultured S. aureus strains was 4.0, whereas the lowest pH values that supported growth and SE production in anaerobic cultures were 4.6 and 5.3 [START_REF] Smith | Effect of Food Environment on Staphylococcal Enterotoxin Synthesis: A Review[END_REF]. Other important parameters influencing the response of S. aureus to pH are the size of inoculum, the type of growth medium, the NaCl concentration (aw), the temperature, and the atmosphere [START_REF] Genigeorgis | Present state of knowledge on staphylococcal intoxication[END_REF]. Most S. aureus strains tested produced detectable amounts of SE aerobically at a pH of 5.1. However, in anaerobic conditions most strains failed to produce detectable SE below pH 5.7 [START_REF] Bergdoll | Staphylococcus aureus in[END_REF][START_REF] Smith | Effect of Food Environment on Staphylococcal Enterotoxin Synthesis: A Review[END_REF][START_REF] Tatini | Influence of food environments on growth of Staphylococcus aureus and production of various enterotoxins[END_REF].

Redox potential

S. aureus is a facultative anaerobic bacterium, which grows best in the presence of oxygen.

Under anaerobic conditions, however, growth is much slower, and even after several days, cell numbers do not reach those attained under aerobic conditions [START_REF] Hennekinne | Staphylococcus aureus as a Leading Cause of Foodborne Outbreaks Worldwide, Staphylococcus aureus[END_REF]. Thus, aerated cultures produced approximately tenfold more SEB than cultures incubated in an atmosphere of 95% N 2+ 5% CO2 [START_REF] Hennekinne | Staphylococcus aureus as a Leading Cause of Foodborne Outbreaks Worldwide, Staphylococcus aureus[END_REF]. The level of dissolved oxygen plays a very important role [START_REF] Bergdoll | Staphylococcus aureus in[END_REF][START_REF] Genigeorgis | Present state of knowledge on staphylococcal intoxication[END_REF]. Under strict anaerobic conditions, the growth of S. aureus was slower as when cultivated aerobically. In broth incubated at 37°C the anaerobic generation time was 80 min compared with 35 min for aerobic culture. With slower anaerobic growth, relatively fewer SEA was produced than under aerobic conditions, but in both cases toxin was detected after 120 min of incubation [START_REF] Belay | Staphylococcus aureus growth and enterotoxin A production in an anaerobic environment[END_REF].

Temperature

S. aureus grows between 7 and 48 °C, temperature being optimal at around 37 °C. The effect of temperature depends on the strain tested and on the type of the growth medium. In a study [START_REF] Schmitt | Temperature limits of growth, TNase and enterotoxin production of Staphylococcus aureus strains isolated from foods[END_REF] using 77 strains isolated from different foods, the optimum growth temperature generally did not vary much within the range of 35-40 °C. The minimum growth temperatures were irregularly distributed between 7 and 13 °C and the maximum between 40 and 48°C. The minimum temperatures for SE production varied quite irregularly over a broad range between 15 and 38°C and the maximum temperatures from 35 to 45 °C. SE formation at 10 °C was reported by [START_REF] Tatini | Influence of food environments on growth of Staphylococcus aureus and production of various enterotoxins[END_REF] without indicating the detailed experimental conditions.

One of the most effective measures for inactivating S. aureus in food is heating. The bacterium is killed in milk if proper heat treatment is applied [START_REF] Hennekinne | Staphylococcus aureus as a Leading Cause of Foodborne Outbreaks Worldwide, Staphylococcus aureus[END_REF]. S. aureus was completely inactivated in milk after application of the following temperature/time conditions: 57.2 °C/80 min, 60.0 °C/24 min, 62.8 °C/6.8 min, 65.6 °C/1.9 min, and 71.7 °C/0.14 min [START_REF] Bergdoll | Staphylococcus aureus in[END_REF]. In the case of heat inactivation in other dairy products, however, one should keep in mind that staphylococci probably become more heat resistant as the aw is lowered until at an aw between 0.70 and 0.80, resistance begins to decline [START_REF] Troller | INFLUENCE OF WATER ACTIVITY ON GROWTH AND ENTEROTOXIN FORMATION BY Staphylococcus aureus IN FOODS[END_REF].

Nutritional factors and bacterial antagonism

Growth of S. aureus and SE production is also influenced by nutritional factors. S. aureus does not grow well in the presence of a competitive flora [START_REF] Hennekinne | Staphylococcus aureus as a Leading Cause of Foodborne Outbreaks Worldwide, Staphylococcus aureus[END_REF]. Its inhibition is mainly because of acidic products, lowering the pH, production of H2O2, or other inhibitory substances such as antibiotics, volatile compounds, or nutritional competition [START_REF] Genigeorgis | Present state of knowledge on staphylococcal intoxication[END_REF][START_REF] Haines | Effect of selected lactic acid bacteria on growth of Staphylococcus aureus and production of enterotoxin[END_REF]. Important factors affecting the degree of inhibition are the ratio of the numbers of competitors to the number of S. aureus as well as the temperature [START_REF] Genigeorgis | Present state of knowledge on staphylococcal intoxication[END_REF][START_REF] Smith | Effect of Food Environment on Staphylococcal Enterotoxin Synthesis: A Review[END_REF]. Starter cultures used in the production of fermented milk products such as cheese, yoghurt, buttermilk, and others can effectively prevent growth of S. aureus and SE formation. In the case of a failure of these cultures, however, the pathogen will not be inhibited and the product may be hazardous [START_REF] Hennekinne | Staphylococcus aureus as a Leading Cause of Foodborne Outbreaks Worldwide, Staphylococcus aureus[END_REF].

Legal regulations

In food industry, food safety is ensured by preventative measures adapted to the risks linked to a specific product. These measures rely on the principles of good hygienic practices (GHP)

as well as the Hazard Analysis and Critical Control Point (HACCP) system. HACCP was originally established by the Pillsbury company, NASA, and the US army to ensure the safety of food products intended for consumption in space [START_REF] Bauman | The origin and concept of HACCP[END_REF]. Nowadays, all food business operators, except the primary producers, must implement HACCP. Good hygienic practices apply to all food business operators, including primary producers. criteria indicate if the production process is performed in a good hygienic manner. These criteria define maximum cell density levels of coagulase-positive staphylococci permitted in food, and they apply during or at the end of the manufacturing process, depending on the food category. Food safety criteria define the acceptability of a foodstuff in terms of its microbiological safety, and they apply during the shelf life of a foodstuff. These criteria define that SEs must not be detected in 25 g of food in any of the sample units. Microbiological criteria for S. aureus counts and enterotoxin detection in foodstuffs are essential to ensure food safety.

However, there are several restrictions to these criteria. Firstly, the number of S. aureus cells is not always a good indicator for the presence of enterotoxins since not all S. aureus strains are enterotoxigenic or express enterotoxins. In addition, even if most S. aureus cells were destroyed e.g. by heat treatment, the heat resistant enterotoxins might still be biologically active and could cause food poisoning. According to regulation an enterotoxin should only be analysed if values >10 5 CFU/g are detected, recent studied show that even in samples with lower concentrations, enterotoxins could be detected (Johler, Weder, et al., 2015;Schwendimann, Berger, et al., 2020).

Secondly, detection of enterotoxins is complex and standard detection methods are limited to classical enterotoxins (SEA-SEE). This is particularly critical since non-classical enterotoxins seem to be involved in foodborne outbreaks (see 2.2.6). In addition the regulation says that enterotoxins have not to be detected in 25 g, without specifying which enterotoxin.

Last but not least according to EC No 852/2004 (EC 2004) and HyV ( 2016) staphylococcal tests of cheese must be conducted at the time when it is assumed that the concentration will be highest. For hard and extra-hard cheese, this is usually before the process step "scalding" starts.

Staphylococcal enterotoxin in cheese

To link the different topics mentioned in the previous chapters and to give an outlook over practical applications of the scientific results, a special sight is given to the topic of staphylococcal enterotoxins in cheese.

The production of cheese and other milk products is culturally and economically particularly important in France and Switzerland. In consequence the fact that cheese, especially fresh and soft cheese made from raw milk, are still involved in SFPO remains an important topic in these countries (Cremonesi et al., 2007;[START_REF] Food Safety | The European Union One Health 2018 Zoonoses Report[END_REF]. The enterotoxins in cheese often originates from raw milk from cows with Staphylococcus aureus mastitis (Schmid et al., 2009). However, poor food safety practices and poor personal hygiene can also be a source of contamination (Hennekinne et al., 2012).

In the alpine region, especially in the case when the contamination with S. aureus happens because of cow's mastitis, two genotypes seem to be predominant: genotype B (GTB) and genotype C (GTC), representing 81% of the isolates (Fournier et al., 2008). In the case of S.

aureus GTB, up to 87% of cows per herd showed intramammary infection, therefore, S. aureus GTB is a contagious mastitis pathogen, which spreads easily among cows (Fournier et al., 2008;Graber et al., 2009;van den Borne et al., 2017). In contrast, S. aureus GTC and most of the other genotypes were detected only in individual cows (Cremonesi et al., 2007;Fournier et al., 2008;Graber et al., 2009).

Staphylococcal genotypes are highly associated with virulence gene patterns (Cosandey et al., 2016;Fournier et al., 2008). Various enterotoxin genes, including sea, sed, sej, and ser, have been identified in S. aureus GTB (Cosandey et al., 2016). This genotype was also shown to persist along the food chain and its enterotoxin genes were expressed and linked to foodborne poisoning outbreaks involving raw milk cheese (Hummerjohann et al., 2014;Kummel et al., 2016). In contrast, S. aureus GTC was typically positive for sec, seg, sei, and tst, (Fournier et al., 2008;Graber et al., 2009) and was not linked to outbreaks traced to raw milk cheese until now (Hummerjohann et al., 2014).

In cheese production, growth of S. aureus can occur prior to processing, as the raw milk used in production may be stored at 8-18° C for up to 24 h before use. During cheese production, temperature, pH, competitive pressure from starter flora, and lactose starvation influence the growth of S. aureus and enterotoxin formation. Salt stress may also play a role in S. aureus growth and enterotoxin formation, although in hard and semi-hard Swiss cheeses, CPS counts start to decline during pressing or a few days after brining when the salt concentration in the cheese is still low [START_REF] Bachmann | Verhalten von potentiell humanpathogenen Bakterien der Arten Aeromonas hydrophila, Campylobacterjejuni, Escherichia coli, Pseudomonas aeruginosa, Salmonella spp., Staphylococcus aureus und Yersinia enterocolitica in Hartund Halbhartkäse aus Rohmilch[END_REF]. Interactions between these parameters may enhance enterotoxin production (Duquenne et al., 2016;[START_REF] Jakobsen | Staphylococcus aureus and Listeria monocytogenes in Norwegian raw milk cheese production[END_REF]Walcher et al., 2014).

Specific studies were performed in the last years regarding the survival and growing of S.

aureus as well as its enterotoxin production. Duquenne et al. (2016) reported that temperature (between 32 and 38° C) and time (15 to 45 min) were key parameters in controlling SE production during the cheese-production process. On the other side Schwendimann et al.

(2020) reported the presence of enterotoxin in cheese containing 10 3 CFU/g (sample taken before salting) and after applying a scalding temperature of 56 °C. Other studies also show how bacteria like Lactococcus lactis (commonly used as starter culture) if added to milk may affect S. aureus growth and viability (Cretenet et al., 2011).

Methods for characterisation of S. aureus, their enterotoxin expression and protein production 2.4.1 Real-time PCR

The development of real-time PCR took place in 1992 [START_REF] Higuchi | Simultaneous amplification and detection of specific DNA sequences[END_REF]. Real-time PCR is able to measure the accumulation of the amplicon instead of making an end point detection as a normal PCR does [START_REF] Josefsen | Istrumentation and fluorescent chemistries used in qPCR[END_REF]. The advantages of real-time PCR are its sensitivity and specificity but also the short duration of the analysis, the reduction of carryover contamination and the reduction of the use of toxic substances like ethidium bromide [START_REF] Josefsen | Istrumentation and fluorescent chemistries used in qPCR[END_REF].

In real-time PCR a fluorescent reporter is introduced that binds to the PCR amplicon and reports its presence. Its presence is expressed by the fluorescence of the dyes or the probes.

This fluorescence is proportional to the amount of product formed and it is monitored during the whole run [START_REF] Kubista | The real-time polymerase chain reaction[END_REF].

In the initial cycles the signal of the fluorescence is weak and cannot be distinguished from the background. If the target DNA is present the amount of fluorescence accumulates in subsequent reaction cycles and a signal develops that increases exponentially, until it reaches saturation [START_REF] Kubista | The real-time polymerase chain reaction[END_REF].

The time that is needed to reach a particular threshold fluorescence level differs depending on the amount of target sequence present at the beginning of the reaction. The number of cycles required to reach the threshold is called Ct-Value. The definition of the threshold is either chosen by the applied software, based on different methods and algorithms, or by the user (see Figure 17) [START_REF] Josefsen | Istrumentation and fluorescent chemistries used in qPCR[END_REF][START_REF] Kubista | The real-time polymerase chain reaction[END_REF]. A variety of different DNA binding dyes and probes chemistry was developed for real-time PCR with the goal to provoque a change in emission of fluorescence during accumulation of the amplicon [START_REF] Josefsen | Istrumentation and fluorescent chemistries used in qPCR[END_REF]. One of the DNA binding dyes is Sybr Green. Sybr Green is an asymmetric cyanine which becomes fluorescent upon intercalating into DNA [START_REF] Kubista | The real-time polymerase chain reaction[END_REF]. The most widely used probe chemistry is the TaqMan® probe. This is a linear targetspecific probe that was designed to complement the sequence in between the forward and the reverse primers. These probes are labelled at the 5' end with a fluorescent reporter dye and at the 3' end with a quencher. When the primers are extended and the probe is encountered, part of the probe will be hydrolysed by the 5'-3' exonuclease activity of the DNA polymerase, separating the reporting dye from the quencher and allowing the emission of fluorescence [START_REF] Josefsen | Istrumentation and fluorescent chemistries used in qPCR[END_REF].

Real-time PCR also found its application in identification and characterisation of S. aureus.

Methods were developed for detection of S. aureus in milk by detecting the nuc gene [START_REF] Graber | Development of a highly sensitive and specific assay to detect Staphylococcus aureus in bovine mastitic milk[END_REF] or for detection of the specific genotype B S. aureus [START_REF] Sartori | Development of a new real-time quantitative PCR assay for the detection of Staphylococcus aureus genotype B in cow milk, targeting the new gene adlb[END_REF]. For the detection of the staphylococcal enterotoxin genes also numerous studies were published [START_REF] Bania | Distribution of newly described enterotoxin-like genes in Staphylococcus aureus from food[END_REF][START_REF] Boss | Mastitis diagnostics: quantitative PCR for Staphylococcus aureus genotype B in bulk tank milk[END_REF][START_REF] Chieffi | Novel insights into the enterotoxigenic potential and genomic background of Staphylococcus aureus isolated from raw milk[END_REF]

Combined methods: PCR and sequencing

Combined methods are used in S. aureus analysis for subtyping the different strains. Multiple methods are available for this purpose. As these methods are not based on the detection of a single gene or genetic sequence, real-time PCR cannot be applied. These methods are often a combination of an end-point PCR and a consequent sequencing of some specific loci. In this chapter selected methods are presented.

MLST

Multi-locus sequence typing (MLST) is currently considered to be one of the most popular typing methods to study the molecular evolution of S. aureus [START_REF] Deurenberg | The evolution of Staphylococcus aureus[END_REF][START_REF] Feng | Evolution and pathogenesis of Staphylococcus aureus: lessons learned from genotyping and comparative genomics[END_REF]. It is based on the sequence analysis of fragments of seven S. aureus housekeeping genes (arcC, aroE, glpF, gmk, pta, tpi, and yqiL), which are about 500 bp in length [START_REF] Enright | Multilocus sequence typing for characterization of methicillin-resistant and methicillin-susceptible clones of Staphylococcus aureus[END_REF]. The alleles resulting from the analysis of these genes build an allelic profile of the strain, which is also defined as sequence type (ST). In the pubMLST database [START_REF] Jolley | Open-access bacterial population genomics: BIGSdb software, the PubMLST.org website and their applications[END_REF], the sequence types (STs) from MLST results can be used to allocate each strain to a clonal complex (CC). Clonal complex are suitable to study the evolutionary events within a S. aureus population [START_REF] Deurenberg | The evolution of Staphylococcus aureus[END_REF].

Genotyping

Ribosomal spacer PCR (RS-PCR) was originally described by [START_REF] Jensen | Rapid identification of bacteria on the basis of polymerase chain reaction-amplified ribosomal DNA spacer polymorphisms[END_REF] as a rapid technique for the identification of pathogenic bacteria belonging to the genera Listeria, Staphylococcus, Salmonella, and other related species. In particular, strains of the genus Staphylococcus showed most significant intra-and interspecies variations in their spacer amplification products [START_REF] Jensen | Rapid identification of bacteria on the basis of polymerase chain reaction-amplified ribosomal DNA spacer polymorphisms[END_REF]. Based on this, Fournier et al. (2008) developed a rapid genotyping method for bovine S. aureus strains, characterized by high discriminatory power. The procedure is based on PCR amplification of the intergenic spacer region between the 16S and the 23S genes of the rRNA genetic loci, using a unified set of primers (Fournier et al., 2008;[START_REF] Jensen | Rapid identification of bacteria on the basis of polymerase chain reaction-amplified ribosomal DNA spacer polymorphisms[END_REF]. The high variability in sequence and length characterizing this intergenic region enables the accurate discrimination of different subtypes of S. aureus (Fournier et al., 2008). After PCR amplification, amplicons are separated according to their size using a miniaturized electrophoresis system (DNA 7500 Chip, Agilent Technologies) resulting in a plot of corresponding peaks (electropherogramm), which can be translated into a pseudogel by a particular software. Results are then evaluated on a personal computer and genotypes are defined by calculating the corresponding Mahalanobis distance of informative peak sizes and comparing it to those of the reference strains [START_REF] Syring | Bovine mastitis: the diagnostic properties of a PCR-based assay to monitor the Staphylococcus aureus genotype B status of a herd, using bulk tank milk[END_REF].

Spa Typing

The spa-typing method can be used for both molecular evolution studies and the investigation of hospital outbreaks of S. aureus [START_REF] Deurenberg | The evolution of Staphylococcus aureus[END_REF]. The method relies on sequencing of the polymorphic X region of the gene encoding the protein A of S aureus (spa), which mainly consists of repeats of 24 bp in length [START_REF] Deurenberg | The evolution of Staphylococcus aureus[END_REF]. The spa gene variability can be the result of spontaneous mutations, as well as deletions or duplications occurring within the repeats [START_REF] Deurenberg | The evolution of Staphylococcus aureus[END_REF][START_REF] Harmsen | Typing of methicillin-resistant Staphylococcus aureus in a university hospital setting by using novel software for spa repeat determination and database management[END_REF]. The Ridom StaphType software is used for data analysis and after a quality control of the constructed consensus sequence, spa-types are deduced from the order of specific repeats, to which alpha-numerical codes are assigned from the software as described by [START_REF] Harmsen | Typing of methicillin-resistant Staphylococcus aureus in a university hospital setting by using novel software for spa repeat determination and database management[END_REF].

Gene expression measurement (RT-qPCR)

For analysis of the expression of a gene, reverse transcriptase real-time PCR (RT-qPCR) is the ideal tool to investigate the transcriptome on RNA level [START_REF] Schnell | Theoretical Description of the Polymerase Chain Reaction[END_REF]. Most gene expression studies quantify mRNAs, but research on other RNA classes such as noncoding RNAs is also evolving rapidly. The first practical real-time PCR technology, the 5'nuclease assay, was established in 1993 and combined the exponential PCR amplification of a specific transcript with the monitoring of newly synthesized DNA in each performed PCR cycle. It was the most sensitive method for the detection and quantification of RNA gene expression levels, in particular for low abundant transcripts [START_REF] Schnell | Theoretical Description of the Polymerase Chain Reaction[END_REF].This method allowed RNA quantification from tissue samples with minor RNA concentrations, and the detection of small changes in mRNA or microRNA expression levels. While real-time RT-PCR has tremendous potential for analytical and quantitative applications, a comprehensive understanding of its underlying principles is important. The fidelity of RT-qPCR is associated with its true specificity, sensitivity, reproducibility, robustness and if performed MIQE compliant as a fully reliable quantitative method. It suffers from the problems inherent in the two single steps, the RT and the PCR, itself [START_REF] Pfaffl | Nucleic Acids -RNA Identification and Quantification via RT-qPCR[END_REF].

For RT-qPCR different single steps are necessary (Figure 18), these are described on the following pages.

RNA extraction

RNA extraction is the first step in RT-qPCR. In this step the total RNA is extracted from the samples. In order to do so, different commercially available kits can be applied. In particular DNA rich samples an additional Dnase step needs to be performed, to eliminate DNA from the sample [START_REF] Taylor | A practical approach to RT-qPCR-Publishing data that conform to the MIQE guidelines[END_REF].

The integrity of the purified RNA, after RNA extraction, is critical to all gene expression analysis and profiling techniques. For successful and reliable diagnostical use, RT-PCR needs high quality, DNA-free, and non-degraded RNA [START_REF] Becker | mRNA and microRNA quality control for RT-qPCR analysis[END_REF]. Accurate quantification and quality assessment of the starting RNA sample is particularly important for absolute quantification methods that normalize specific mRNA expression levels against total RNA ("molecules/g total RNA" or "transcript concentrations/g total RNA").

In order to check the purity of RNA according to the applied MIQE guideline [START_REF] Taylor | The state of RT-quantitative PCR: firsthand observations of implementation of minimum information for the publication of quantitative real-time PCR experiments (MIQE)[END_REF] RNA integrity (RIN > 7.0) and RNA purity (A260/A280 > 1.8) should be measured.

Reverse transcription

The extracted RNA is then transcribed into complementary DNA (cDNA). These steps can be combined with qPCR (one step) or performed singularly (two steps), depending on the requirements of the user (Figure 19). For the reverse transcription different types of primers can be used, again the application is depending on the user's requirements, following options are possible: Oligo(dT)s or anchored oligo(dT)s), random primers and Sequence Specific Primers [START_REF] Taylor | The state of RT-quantitative PCR: firsthand observations of implementation of minimum information for the publication of quantitative real-time PCR experiments (MIQE)[END_REF]. To confirm the absence of residual DNA, a minus-RT should be included in the experimental setting, as recommended by the MIQE guidelines. Additionally, it may be necessary to treat the RNA sample with commercially-available RNAse-free DNAse to get rid of any unwanted residual cellular DNA [START_REF] Taylor | The state of RT-quantitative PCR: firsthand observations of implementation of minimum information for the publication of quantitative real-time PCR experiments (MIQE)[END_REF].

The RT step is the major source of variability in a real-time RT-PCR experiment, and the specific reaction conditions must be optimized for each RT enzyme. It cannot be assumed that different reaction setups have an identical cDNA synthesis efficiency. Therefore, the result can be highly variable during multiple RT reactions (Ståhlberg, Kubista, et al., 2004;[START_REF] Ståhlberg | Comparison of Reverse Transcriptases in Gene Expression Analysis[END_REF]. According to literature to circumvent the high inter-assay variations in RT, target gene-unspecific primers, for example, random-hexamer, octamer, or -decamer primers should be used to synthesize the cDNA pool [START_REF] Pfaffl | Nucleic Acids -RNA Identification and Quantification via RT-qPCR[END_REF].

Real-time PCR

Real-time PCR has already been described in chapter 2.4.1. For the application in RT-qPCR some specific considerations need to be considered. The amount of amplified target is directly proportional to the number of target molecules in the sample, but only during the "exponential amplifying phase" of the PCR reaction. Hence the key factor in the quantitative ability of RT-qPCR is that it measures the product of the target gene only during this phase. Since data acquisition and analysis are performed in the same tube, this increases sample throughput, reduces carryover contamination and removes post-PCR processing as a potential source of error. In contrast, there is no direct relation of DNA input to amplified target during the plateau phase of the PCR; therefore, classical RT-PCR assays have to be stopped in late exponential and at latest in the linear phase. The exponential range of amplification has to be empirically determined for each transcript by amplifying equivalent amounts of cDNA over various PCR cycles or by amplifying dilutions of cDNA over the same number of PCR cycles [START_REF] Pfaffl | Nucleic Acids -RNA Identification and Quantification via RT-qPCR[END_REF].

Quantification strategies

Two general quantification strategies can be performed in RT-qPCR: The levels of expressed genes may be measured by "absolute quantification" or by "relative quantification" (Figure 20).

Absolute quantification relates the PCR signal to input copy number using a calibration curve, while relative quantification measures the relative change in mRNA or microRNA expression levels [START_REF] Pfaffl | Relative Quantification[END_REF]. The reliability of an absolute real-time RT-PCR assay depends on the condition of identical amplification efficiency for both the native target and the calibration curve in RT reaction and following qPCR. Nowadays, relative quantification is more commonly performed than absolute quantification because there is no need for a calibration curve. It is based on the expression levels of a target gene versus a reference gene, usually a classical housekeeping gene.

Normalization of expression results

The reliability of any relative RT-PCR expression can be improved by including an invariant endogenous control in the assay to correct for sample-to-sample variations in RT-PCR efficiency and errors in sample quantification [START_REF] Pfaffl | Nucleic Acids -RNA Identification and Quantification via RT-qPCR[END_REF]. A biologically meaningful reporting of target mRNA copy numbers requires accurate and relevant normalization to some standard and is strongly recommended [START_REF] Vandesompele | Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes[END_REF][START_REF] Vandesompele | Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes[END_REF]. But the quality of normalized quantitative expression data cannot be better than the quality of the normalizer itself. Any variation in the normalizer will obscure real changes and produce artificial changes. Real-time RT-PCR-specific errors in the quantification of RNA transcripts are easily compounded with any variation in the amount of starting material between the samples, for example, caused by sample-to-sample variation, variation in RNA integrity, differences in RT efficiency and cDNA sample loading variation [START_REF] Taylor | The state of RT-quantitative PCR: firsthand observations of implementation of minimum information for the publication of quantitative real-time PCR experiments (MIQE)[END_REF]. This is especially relevant when the samples have been obtained from different individuals, different tissues and different time courses and will result in the misinterpretation of the derived expression profile of the target genes. Therefore, normalization of target gene expression levels must be performed to compensate intra-and inter-RT-qPCR variations. Data normalization can be carried out against an endogenous unregulated reference gene transcript or against total cellular DNA or RNA content (molecules/g total DNA/RNA and concentrations/g total DNA/RNA). Normalization based on the amount of total cellular RNA content is increasingly used, but little is known about the total RNA content of cells or even about the mRNA concentrations. The content per cell or per gram of tissue may vary in different tissues (in vivo) and cell cultures (in vitro), between individuals and under different experimental conditions [START_REF] Pfaffl | Nucleic Acids -RNA Identification and Quantification via RT-qPCR[END_REF].

Application in measurement of the staphylococcal enterotoxin expression

RT qPCR has been widely applied for detection of enterotoxin gene expression. It has been used for analysing the expression of se under different stress parameters [START_REF] Sihto | Growth behavior and temporal enterotoxin D expression of Staphylococcus aureus strains under glucose and lactic acid stress[END_REF][START_REF] Sihto | Temporal expression of the staphylococcal enterotoxin D gene under NaCl stress conditions[END_REF][START_REF] Wallin-Carlquist | Acetic acid increases the phage-encoded enterotoxin A expression in Staphylococcus aureus[END_REF][START_REF] Zeaki | Evaluation of Potential Effects of NaCl and Sorbic Acid on Staphylococcal Enterotoxin A Formation[END_REF] or by addition of mitomycin [START_REF] Cao | Elevated enterotoxin A expression and formation in Staphylococcus aureus and its association with prophage induction[END_REF]. These studies were mostly performed on classical enterotoxins, even they were also performed for the non-classical enterotoxins [START_REF] Derzelle | Differential temporal expression of the staphylococcal enterotoxins genes during cell growth[END_REF][START_REF] Omoe | Detection of seg, seh, and sei genes in Staphylococcus aureus isolates and determination of the enterotoxin productivities of S. aureus isolates Harboring seg, seh, or sei genes[END_REF][START_REF] Sato'o | Positive Regulation of Staphylococcal Enterotoxin H by Rot (Repressor of Toxin) Protein and Its Importance in Clonal Complex 81 Subtype 1 Lineage-Related Food Poisoning[END_REF] Gene expression has also already be performed in different food matrices like pork sausages [START_REF] Zeaki | Assessment of high and low enterotoxin A producing Staphylococcus aureus strains on pork sausage[END_REF], boiled ham [START_REF] Susilo | Reduced Enterotoxin D Formation on Boiled Ham in Staphylococcus Aureus Deltaagr Mutant[END_REF], meat product [START_REF] Sato'o | Positive Regulation of Staphylococcal Enterotoxin H by Rot (Repressor of Toxin) Protein and Its Importance in Clonal Complex 81 Subtype 1 Lineage-Related Food Poisoning[END_REF], milk [START_REF] Valihrach | Expression and production of staphylococcal enterotoxin C is substantially reduced in milk[END_REF], and cheese (Duquenne et al., 2016;[START_REF] Duquenne | Tool for quantification of staphylococcal enterotoxin gene expression in cheese[END_REF].

Whole genome sequencing

Genome sequencing has started in the 1970-1980 when manual methods were used like Sanger and Maxam Gilbert sequencing. With these techniques whole bacteriophages and animal viral genomes were sequenced [START_REF] Alberts | Molecular Biology of the Cell[END_REF]. In the 90' faster and automated techniques allowed sequencing of bacteria and eukaryota. The first bacteria to be fully sequenced was Haemophilus influenza in 1995 [START_REF] Fleischmann | Whole-genome random sequencing and assembly of Haemophilus influenzae Rd[END_REF].

Microbial genome sequencing has become mainstream in the field of food microbiology due to the increasing affordability and improvements in the speed of sequencing and quality of the data. This was a consequence of the advancements in sequencing technologies collectively known as next generation sequencing (NGS). NGS encompasses both massively parallel and single-molecule sequencing which provides short and long sequencing reads, respectively.

Short-read sequencing is highly accurate and produces read lengths of 100-300 bp which are then assembled into incomplete or so called, draft genomes [START_REF] Jagadeesan | The use of next generation sequencing for improving food safety: Translation into practice[END_REF].

Complete genomes cannot be generated from the short reads obtained in a single sequence run due to difficulties in assembling repetitive regions and large genomic rearrangements such as insertions, deletions and inversions [START_REF] Jagadeesan | The use of next generation sequencing for improving food safety: Translation into practice[END_REF]. For many applications, including comparative genomics and phylogeny, this is not an issue but where complete genomes are required and for determining complex genomic regions, longer reads are necessary. Long-read sequencing produces reads from 10 to 50 Kb in length, but this is at the cost of higher error rates [START_REF] Loman | Twenty years of bacterial genome sequencing[END_REF]. Currently, microbial DNA sequencing can be performed on a variety of platforms Table 9. WGS of microbial pathogens has been introduced into public health surveillance relatively rapidly compared to previous methodological advancements, with reports of its use from early adopters from 2011 onward [START_REF] Lienau | Identification of a Salmonellosis Outbreak by Means of Molecular Sequencing[END_REF][START_REF] Rall | Routine Use of Microbial Whole Genome Sequencing in Diagnostic and Public Health Microbiology[END_REF]. First it was used in retrospective analysis of foodborne outbreaks. Nowadays it becomes a powerful tool for prospective analysis and surveillance of bacterial foodborne pathogens [START_REF] Allard | Genomics of foodborne pathogens for microbial food safety[END_REF][START_REF] Ashton | Identification ofSalmonellafor public health surveillance using whole genome sequencing[END_REF][START_REF] Jackson | Implementation of Nationwide Real-time Whole-genome Sequencing to Enhance Listeriosis Outbreak Detection and Investigation[END_REF][START_REF] Moura | Whole genome-based population biology and epidemiological surveillance of Listeria monocytogenes[END_REF]. WGS is being introduced as a replacement technology, i.e. it will replace most current identification and characterisation methods in the microbiology laboratory such as serotyping, virulence profiling, antimicrobial resistance determination and previous molecular typing methods. In a public health setting replacing the plethora of traditional microbiological identification and typing methods with a single efficient analytical WGS workflow makes implementation cost-effective as well as more accurate. Actionable data on public health can be collected faster than previously [START_REF] Grant | Implementing pathogen genomics: a case study[END_REF].

Analysis of WGS data by either approach is a complex process in which multiple steps are combined to produce final results, such as SNP or allele matrices and phylogenetic trees [START_REF] Timme | Benchmark datasets for phylogenomic pipeline validation, applications for foodborne pathogen surveillance[END_REF]. The large amount of data generated in WGS brings challenges for its analysis [START_REF] Deurenberg | Application of next generation sequencing in clinical microbiology and infection prevention[END_REF][START_REF] Wyres | WGS Analysis and Interpretation in Clinical and Public Health Microbiology Laboratories: What Are the Requirements and How Do Existing Tools Compare?[END_REF]. This has led to multiple software solutions being developed, mainly through academic endeavours, which in general require specialised knowledge and expertise to be deployed and operated. More recently commercially developed software have become available, bringing a user-friendly interface, allowing non-bioinformatics experts, with the appropriate training in both bioinformatics software and final WGS result interpretation, to conduct analyses. The commercial software may be expensive but since limited bioinformatics expertise is needed, it may nevertheless be a more cost-efficient solution for many users in food industry. Two main applications to analyse genomic data to determine the relatedness between strains, namely SNP-based and the gene by gene-based approaches (such as wgMLST and cgMLST) are available. In the SNP-based approach, sequencing reads are aligned or mapped to a known sequenced reference genome, and the nucleotide differences in both coding and non-coding regions determined [START_REF] Davis | CFSAN SNP Pipeline: an automated method for constructing SNP matrices from next-generation sequence data[END_REF]. For each isolate, every SNP relative to the reference genome is recorded and then used to quantify the genetic relatedness between strains. CgMLST and wgMLST are an extension to traditional 7-loci multi-locus sequence typing (MLST), the genes in either a defined core genome (cgMLST) or the whole genome (wgMLST), which includes more variable accessory genes, are compared against a reference database of all known gene variants (alleles) for a particular species. CgMLST provides highly detailed phylogenetically relevant information about the genetic relatedness of a species. On the other side wgMLST provides even more discrimination than cgMLST and this can be valuable for cluster investigations to discriminate between closely related isolates. The genetic variation detected by SNP or gene-by-gene analysis can be used to infer phylogenetic relationships between bacterial isolates usually displayed in the form of a phylogenetic tree. The tree represents the calculated evolutionary model (obtained using different possible tree inference algorithms such as parsimony, maximum likelihood, and Bayesian or distance methods) of the isolates as a series of branches from the root or common ancestor. The isolates clustered together near the leaves of the tree are more closely related than other isolates elsewhere in the tree [START_REF] Jagadeesan | The use of next generation sequencing for improving food safety: Translation into practice[END_REF].

About application of whole genome sequencing in S. aureus can be read in chapter 2.1.2.

Immunoassays

Immunoassay is an analytical method that is widely applied in many fields, including pharmaceutical analysis, toxicological analysis, bioanalysis, clinical chemistry, and environmental analysis based on specific recognition between antigens and antibodies.

Immunoassays are extremely sensitive and specific, rapid to operate, and can be used to detect SEs in complex samples without extensive pre-treatment. Combining antibodies as a recognition component with an appropriate transducer formed biosensor called immunosensor. In immunosensor, sensing elements play an important role in the detection process and are basic devices giving an output in the form of measurable energy that is correlated with the input quantity. According to the applied transduction patterns, these sensors can be classified into three main types: (1) optical detection techniques, [START_REF] Bachmann | The fate of potentially pathogenic bacteria in Swiss hard and semihard cheeses made from raw milk[END_REF] electrochemical detection techniques and (3) mass detection techniques (Wu et al., 2016).

Colorimetric immunoassays are belonging to the optical detection techniques and are the most common ones. They determine analytes by comparing or measuring the absorbance of a colourful substance. The transducer moiety is a key component of colorimetric immunoassays that affects performance with respect to sensitivity, specificity, response time, and the signalto-noise ratio, due to its function of translating the detecting behaviour into light absorption ranging from 390 to 750 nm, characterized by an eye-sensitive colour change. Enzyme-linked immunosorbent assay (ELISA) is a widely used colorimetric method. ELISAs are commonly performed by immobilizing artificial antigens or capturing antibodies on plastic supports. The antigen captured by the antibody support can be detected either using an enzyme-labelled antibody that is specific for the same determinant as the capture antibody or by an enzymelabelled antibody recognizing a different epitope on the captured, multivalent antigen.

Quantification of antigens is achieved by monitoring the cleavage of the chromogenic substrate (e.g., 3,31,5,51-tetramethylbenzidine (TMB)) by the enzyme (e.g., horseradish peroxidase (HRP)), which produces a blue metabolite for signal detection. These methods have many advantages, such as being easy to use, specific, and applicable for high throughput screening.

SEs are routinely assayed immunologically by ELISA (Wu et al., 2016).

By looking in literature different immunological assays were developed in the last year for the non-classical enterotoxins like for SEG [START_REF] Nagaraj | Development of IgY based sandwich ELISA for the detection of staphylococcal enterotoxin G (SEG), an egc toxin[END_REF], SEH [START_REF] Schubert | Production of staphylococcal enterotoxins in microbial broth and milk by Staphylococcus aureus strains harboring seh gene[END_REF], SEI (Zhao et al., 2017a), SEK [START_REF] Aguilar | Detection and Measurement of Staphylococcal Enterotoxin-Like K (SEl-K) Secretion by Staphylococcus aureus Clinical Isolates[END_REF], SEM (Zhao et al., 2017b) and SEQ [START_REF] Hu | Biological characteristics of staphylococcal enterotoxin Q and its potential risk for food poisoning[END_REF]. Unfortunately, they are all not commercially available.

Research objectives

Based on the literature search, the research objectives and the type of enterotoxin to study were chosen. Based on these findings egc enterotoxins (seg, sei, sem, sen, seo) were selected to be further studied. This decision was taken based on following facts:

 Egc enterotoxins seem to be present in ~50 % of the S. aureus strains  Emetic activity of seg, sei, sem and seo have been demonstrated

 Few is known about the expression of egc enterotoxins, the way they are regulated and how different stress parameters are influencing their expression  Recent studies seem to show the involvement of egc enterotoxins in foodborne outbreaks

The aim of this study is to develop a prediction model for egc enterotoxin production based on WGS data, as presented in Figure 21: More in detail, the vSaβ (containing the egc) of a large strain collection is analyzed in-silico.

Expression of SE gene systems is then studied by reverse transcription quantitative PCR (RT-qPCR) using different stressors (environmental conditions). These results are then confirmed by and compared with the results obtained with ELISA (for detection of SEG and SEI) and used to set up the model to predict SE expression based on in-silico analysis of WGS data.

In order to achieve this aim, the work was divided into nine different tasks:

1. Search for available strain collections having S. aureus strains originating from foods, SFPOs, environmental and animals. Characterization for their presence of egc.

2. Selection of a subset of these strains and if sequencing data not available, sequencing by WGS.

3. Application of bioinformatic methods to create a genomic scaffold to locate the vSaβ and their arrangement within the genome.

4. Analysis of the egc enterotoxin promoter regions of the different vSaβ.

5.

Based on diversity of vSaβ type, origin and genetic diversity, selection and analysis for their production of SEG and SEI of a subset of strains.

6. Development of a method (RT-qPCR) in order to measure the expression of egc enterotoxins (seg, sei, sem, sen, seo).

7. Based on the results of point 5, selection of different strains and study of the correlation between vSaβ and se expression using RT-qPCR. In addition, evaluation of gene expression of egc enterotoxins under different stress parameters such as NaCl and temperature.

8. Comparison of the results obtained under point 6 with the results obtained using ELISA methods for detection of SEG and SEI.

9. Set up of the prediction model.

Strain collection and available methods

As stated in point 1 of the previous chapter strains from different collections were available from following collections: From all these strains a subset of 75 egc positive strains was chosen. Where information were not available, real-time PCR for detection of the egc enterotoxins was applied. The choice of the strains was based on their diversity, the aim was to reach egc positive strains with the widest possible diversity. In order to do so, following criteria were applied:



Oral communications at international congresses:

 EUNRL Workshop, June 2018

Abstract

Currently only five (SEA-SEE) out of 26 known staphylococcal enterotoxins can be analyzed using commercially available kits.

Six genes (seg, sei, sem, sen, seo, and seu), encoding putative and undetectable enterotoxins, are located on the enterotoxin gene cluster (egc) which is part of the Staphylococcus aureus genomic island vSaβ. These enterotoxins have been described as likely being involved in staphylococcal food poisoning outbreaks.

The aim of the present study was to determine if whole genome data can be used for the prediction of staphylococcal egc enterotoxin production, particularly enterotoxin G (SEG) and enterotoxin I (SEI). For this purpose whole genome sequences of 75 Staphylococcus aureus (S. aureus) strains from different origins (food poisoning outbreaks, human, and animal) were investigated applying bioinformatics methods (phylogenetic analysis using the core genome and different alignments). SEG and SEI expression was tested in vitro using a sandwich ELISA method.

Strains could be allocated to 14 different vSaβ types, each type being associated with a single clonal complex (CC). In addition the vSaβ type and CC were associated with the origin of the strain (human or cattle derived). The amount of SEG and SEI produced also correlated with the vSaβ type and the CC of a strain. The present results show promising indications that the in vitro production of SEG and SEI can be predicted based on the vSaβ type or CC of a strain.

Importance

Beside the infection properties in human and animals, S. aureus can produce different enterotoxins in food. These enterotoxins can cause vomiting and diarrhea, often involving many people. Most of these outbreaks remain undiscovered as detection methods for enterotoxins are only available for a few enterotoxins, but not for the more recently discovered enterotoxins G (SEG) and I (SEI).

In this study we show promising results that in vitro production of SEG and SEI can be predicted based on the whole genome sequencing data of a strain. In addition, this data could be used to find the source (human-or cattle-derived) of an outbreak strain, which is the key for a better understanding of the role SEG and SEI play in foodborne outbreaks caused by S.

aureus.

Résumé (French)

Actuellement, seules cinq (SEA-SEE) des 26 entérotoxines staphylococciques connues peuvent être analysées à l'aide de kits disponibles dans le commerce. can be detected and quantified by commercially available kits [START_REF] Hennekinne | Intralaboratory validation according to the EN ISO 16 140 Standard of the Vidas SET2 detection kit for use in official controls of staphylococcal enterotoxins in milk products[END_REF].

Beside these five well-known SE another 20 have been described recently and some of them were shown to have an emetic activity (SE) and hence, could be involved in SFPOs [START_REF] Benkerroum | Staphylococcal enterotoxins and enterotoxin-like toxins with special reference to dairy products: An overview[END_REF][START_REF] Johler | Further evidence for staphylococcal food poisoning outbreaks caused by egc-encoded enterotoxins[END_REF][START_REF] Langley | Staphylococcal enterotoxin-like X (SElX) is a unique superantigen with functional features of two major families of staphylococcal virulence factors[END_REF][START_REF] Ono | The emetic activity of staphylococcal enterotoxins, SEK, SEL, SEM, SEN and SEO in a small emetic animal model, the house musk shrew[END_REF][START_REF] Ono | Identification and characterization of two novel staphylococcal enterotoxins, types S and T[END_REF][START_REF] Ono | Identification and Characterization of a Novel Staphylococcal Emetic Toxin[END_REF][START_REF] Zhang | Identification and characterization of two novel superantigens among Staphylococcus aureus complex[END_REF]. Enterotoxins for which emetic activity has not yet been proved are considered as staphylococcal enterotoxin like (SEl) proteins. As all SE cannot be detected directly in food, different methods have been applied in the past to better characterize the S. aureus strains involved in food poisoning outbreaks, such as pulsedfield gel electrophoresis typing, PCR for detection of the enterotoxins genes and other methods [START_REF] Chieffi | Novel insights into the enterotoxigenic potential and genomic background of Staphylococcus aureus isolated from raw milk[END_REF][START_REF] Ciupescu | Characterization of Staphylococcus aureus strains and evidence for the involvement of non-classical enterotoxin genes in food poisoning outbreaks[END_REF][START_REF] Fusco | Rapid and reliable identification of Staphylococcus aureus harbouring the enterotoxin gene cluster (egc) and quantitative detection in raw milk by real time PCR[END_REF][START_REF] Kerouanton | Characterization of Staphylococcus aureus strains associated with food poisoning outbreaks in France[END_REF][START_REF] Yan | Characterization of Staphylococcus aureus strains associated with food poisoning in Shenzhen, China[END_REF]. These methods allowed to evaluate the toxigenic profile of strains, or to establish the link between strains and secreted toxins. With the recent advance of whole genome sequencing (WGS), often each strain involved in an outbreak can be sequenced and characterized genetically, opening new doors to the understanding of the role different SE play in SFPO as well as prediction of antimicrobial resistances and infectivity [START_REF] Baba | Genome sequence of Staphylococcus aureus strain Newman and comparative analysis of staphylococcal genomes: polymorphism and evolution of two major pathogenicity islands[END_REF][START_REF] Fitzgerald | Characterization of a putative pathogenicity island from bovine Staphylococcus aureus encoding multiple superantigens[END_REF][START_REF] Gordon | Prediction of Staphylococcus aureus antimicrobial resistance by whole-genome sequencing[END_REF][START_REF] Macori | Genome-Wide Profiling of Enterotoxigenic Staphylococcus aureus Strains Used for the Production of Naturally Contaminated Cheeses[END_REF][START_REF] Manara | Whole-genome epidemiology, characterisation, and phylogenetic reconstruction of Staphylococcus aureus strains in a paediatric hospital[END_REF][START_REF] Price | The usefulness of whole genome sequencing in the management of Staphylococcus aureus infections[END_REF].

Twenty years ago a novel cluster of se genes, the enterotoxin gene cluster (egc), was described containing the so-called "new enterotoxins" seg, sei, sem, sen, seo, and seu [START_REF] Jarraud | egc, a highly prevalent operon of enterotoxin gene, forms a putative nursery of superantigens in Staphylococcus aureus[END_REF][START_REF] Thomas | Staphylococcal enterotoxin-like toxins U2 and V, two new staphylococcal superantigens arising from recombination within the enterotoxin gene cluster[END_REF]. The egc is located on the genomic island vSaβ and is incorporated in the chromosome as a prophage [START_REF] Baba | Genome sequence of Staphylococcus aureus strain Newman and comparative analysis of staphylococcal genomes: polymorphism and evolution of two major pathogenicity islands[END_REF]. Literature suggests that about 50% of S. aureus strains harbour an egc [START_REF] Argudin | Genotypes, exotoxin gene content, and antimicrobial resistance of Staphylococcus aureus strains recovered from foods and food handlers[END_REF][START_REF] Jarraud | egc, a highly prevalent operon of enterotoxin gene, forms a putative nursery of superantigens in Staphylococcus aureus[END_REF][START_REF] Smyth | Superantigen genes encoded by the egc cluster and SaPIbov are predominant among Staphylococcus aureus isolates from cows, goats, sheep, rabbits and poultry[END_REF].

For SEG, SEI, SEM, SEN, and SEO emetic activity has been demonstrated and it appears that some SFPO might be caused by these enterotoxins [START_REF] Johler | Further evidence for staphylococcal food poisoning outbreaks caused by egc-encoded enterotoxins[END_REF][START_REF] Ono | The emetic activity of staphylococcal enterotoxins, SEK, SEL, SEM, SEN and SEO in a small emetic animal model, the house musk shrew[END_REF]. A lot is known on the expression of the classical SE [START_REF] Sihto | Temporal expression of the staphylococcal enterotoxin D gene under NaCl stress conditions[END_REF][START_REF] Valihrach | Expression and production of staphylococcal enterotoxin C is substantially reduced in milk[END_REF][START_REF] Zeaki | Evaluation of Potential Effects of NaCl and Sorbic Acid on Staphylococcal Enterotoxin A Formation[END_REF], yet studies on the expression of the new SE are still very limited (Zhao et al., 2017b). Genetic backbones and regulatory systems of SE genes vary among S.

aureus strains, causing diverse SE expression patterns. Hence, quantities of toxin production vary between strains [START_REF] Sihto | Temporal expression of the staphylococcal enterotoxin D gene under NaCl stress conditions[END_REF][START_REF] Valihrach | Expression and production of staphylococcal enterotoxin C is substantially reduced in milk[END_REF][START_REF] Zeaki | Evaluation of Potential Effects of NaCl and Sorbic Acid on Staphylococcal Enterotoxin A Formation[END_REF].

Due to the lack of information, new methods and tools need to be developed in order to better understand and predict the expression and regulation mechanisms of the new enterotoxins including those of the egc [START_REF] Zeaki | The Role of Regulatory Mechanisms and Environmental Parameters in Staphylococcal Food Poisoning and Resulting Challenges to Risk Assessment[END_REF]. For this reason, the aim of the present study was to determine whether WGS data can be used to predict staphylococcal enterotoxin production of the egc in vitro, particularly of SEG and SEI. These enterotoxins (SEG and SEI)

were chosen because of the fact that they are the only ones (of egc enterotoxins) for which a quantitative method for detection is currently available, allowing a direct link for the corresponding WGS data.

Results

Strain characterization

Multi-locus sequence typing (MLST) of the 75 S. aureus strains, isolated from different sources like food, human, animals and environment, showed that the most frequently found clonal complexes (CC) are CC5 (n = 17), CC20 (n = 15), CC30 (n = 13), and CC705 (n = 11). Followed by CC45, CC22, CC50, and CC9 (6, 3, 2, and 2 strains, respectively). In contrast CC10, CC72, CC121, as well as an unknown CC were detected only once (Table 10).

The strains from the most frequently found CCs (CC5 and CC30) originated from a vast geographical range and were isolated from either human or food. In contrast, the CC20 and CC705 strains, always originated from France, Italy and Switzerland, and were isolated either from dairy products or bovine mastitis (Table 10).

Spa-typing of the 75 strains revealed that in most cases the strains belonging to a single CC were allocated to different spa-types. Perfect agreement between CC and spa-type was found only for CC705 (n=11), where all strains were allocated to t529. For 15 strains spa-typing resulted in an unknown type, of which the majority belonged to CC30 and CC20 (5 and 6

unknown spa-types, respectively).

Besides the egc, the 75 strains also harboured other non-egc SE genes (Table 10). Indeed, from genome assembly, all 26 SE genes were at least once detected in one of the strains. Yet it is noteworthy that the five strains belonging to CC5 often carried additional SE genes such as selx (in 4 strains), sea (in 3 strains), and a plasmid containing sed, sej, and ser (in 2 strains).

Furthermore, CC30 (n= 13) harboured sea in 6 strains and tst (toxic-shock toxin) in 2 strains.

CC705 comprised sec, tst, selx, and sel, whereas CC20 often carried selx and sey (in 14 and

Allocation of the strains to their vSaβ types and diversity of SEG and SEI

In 59 of 75 strains (79%) the vSaβ type could be allocated to an existing one with overall similarities of >90%. For the remaining 16 strains new vSaβ types were defined by numbering continuously from XVI onward (Figure 22), resulting in seven new vSaβ types (XVI-XXII). Three strains were allocated to vSaβ type XVI, two strains to vSaβ type XXI, and six strains to vSaβ type XXII, respectively (Table 10). For the remaining vSaβ types (XVII, XVIII, XIX, XX) only one strain each was found.

The seven newly defined vSaβ types (Figure 22) all contained, in addition to the egc genes, virulence-associated and hypothetical genes. vSaβ types XVII and XVIII carry bacteriocins and serine proteases, whereas vSaβ type XIX was notably (approx. 20'000 bp) longer than the other vSaβ types and carried numerous genes coding for hypothetical proteins. vSaβ type XXII was shorter than all other vSaβ types (approx. 13'000 bp) and did not carry any additional virulence-associated genes besides the egc genes. Within each vSaβ type, an amino acid identity of 100% for each SE was observed. However, SE differences were observed among different vSaβ types (Table 11). Among all strains included in the study, the SEG amino acid similarity varied between 96% and 100%, with a maximum of 9 amino acids difference, compared to strain Mu50 (reference). For SEI the similarity varied between 93% and 100%, with a maximum difference, of 19 amino acids.

Phylogenetic analysis of the core genome

In order to evaluate the evolutionary relationship of S. aureus strains included in the present study, their phylogeny was evaluated based on their core genome. The tree shows a perfect concordance between the phylogenetic clades, CCs, and vSaβ type of the strain (Figure 23).

For vSaβ type IV, XI, XII and XIII a perfect concordance was observed between strains isolated from milk products and animal mastitis can be observed (no human strains harboured these vSaβ types). On the other side, strains harbouring vSaβ type I III and XXII were only found in humans (including infections) and food isolates. No animal strains harboured these vSaβ types. SFPO strains were found in every vSaβ type.

Enterotoxin production

SEG production ranged from below the limit of detection (LOD 0.001 ng/ml) to 4.26 +/-0.78 ng/ml, with a median of 1.17 ng/ml. SEG production below LOD (0.001 ng/ml) was observed for vSaβ IV and XXI. One strain carrying vSaβ III (18SBCL675) showed non-detectable quantities of SEG, whereas the other two strains harbouring vSaβ III had values between 0.26 +/-0.01 and 0.78 +/-0.13 ng/ml. All the other strains showed values between 0.80 +/-0.11 and 4.26 +/-0.78 g/ml. By visual data inspection (Figure 24A), two levels of SEG production can be distinguished: 9 strains that generated low (L) and 23 strains that produced high concentrations (H) of SEG. The median concentration for the L producer was 0 ng/ml (min 0 ng/ml, max 0.26 +/-0.01 ng/ml) and for the H producer 1.42 +/-0.14 ng/ml (min. 0.783 +/-0.13 ng/ml, max 4.26 +/-0.78 ng/ml). The medians differed highly significantly (P < 0.001).

The amount of SEI produced (LOD 0.037 ng/ml) by the strains ranged from 1.06 +/-0.17 ng/ml to 61.43 +/-10.29 ng/ml (median 14.31 ng/ml) (Table 10, Figure 24B). According to their SEI production, strains could again be visually allocated to two different levels: L-producers (producing 1.06 +/-0.17 -3.85 +/-0.99 ng/ml, median 2.22 ng/ml) and H-producers (10.77 +/-1.22-61.43 +/-10.29 ng/ml, median 21.51 ng/ml). The L-strains belonged to the vSaβ types III, IV, and XI, whereas the H-strains belonged to vSaβ types I, XII, XIII, XVI, XVII, XVIII, XIX, XX, XXI, or XXII (P < 0.001, between L and H). To assess a possible relationship between SEG and SEI production, a first robust linear regression (Appendix II, Figure S1-S2) was performed identifying four outliers (G68P, 18SBCL669, Mu50 and 05CEB52). These outliers were not taken into consideration for a A B second, ordinary least square linear regression analysis (Appendix II, Figure S3). This regression was modelled to

[SEI] = 15.49 x [SEG] + 0.63, with R=0.940 (P <0.001), were [] are the SE concentrations in ng/ml.

Discussion

In the present study we demonstrate that SEG and SEI production in vitro can be predicted using genomic data. In fact, there is a strong indications that the amount of these SE produced depends on the vSaβ type. Furthermore, with the analysis and findings described here, it is now possible to infer the origin of an egc containing S. aureus strain (human-derived, cattlederived) that is involved in an SFPO. As the vSaβ type is perfectly linked to the CC of a strain, as shown in the present study and in the previous from Klaui et al. [START_REF] Klaui | Characterization and Comparative Analysis of the Staphylococcus aureus Genomic Island vSabeta: an In Silico Approach[END_REF], the SE production and the origin of the SFPO can also be predicted based on the CC of the strain obtained by MLST, a typing method that is well established nowadays.

Previous studies already demonstrated that different strains can produce different amounts of SE, but in most cases the link to the genome was missing [START_REF] Borst | Phage-associated differences in staphylococcal enterotoxin A gene (sea) expression correlate with sea allele class[END_REF][START_REF] Derzelle | Differential temporal expression of the staphylococcal enterotoxins genes during cell growth[END_REF].

In this study the focus was led on the egc enterotoxins, that, according to previous studies [START_REF] Johler | Further evidence for staphylococcal food poisoning outbreaks caused by egc-encoded enterotoxins[END_REF][START_REF] Song | Molecular Typing and Virulence Gene Profiles of Enterotoxin Gene Cluster (egc)-Positive Staphylococcus aureus Isolates Obtained from Various Food and Clinical Specimens[END_REF][START_REF] Umeda | Molecular and epidemiological characterization of staphylococcal foodborne outbreak of Staphylococcus aureus harboring seg, sei, sem, sen, seo, and selu genes without production of classical enterotoxins[END_REF][START_REF] Yan | Characterization of Staphylococcus aureus strains associated with food poisoning in Shenzhen, China[END_REF], is harboured by about 50% of S. aureus strains. The importance of the egc enterotoxins regarding food safety has been shown by Johler et al. [START_REF] Johler | Further evidence for staphylococcal food poisoning outbreaks caused by egc-encoded enterotoxins[END_REF] who described the probable egc enterotoxins' involvement in foodborne outbreaks. However, strong evidence could not be confirmed, as the enterotoxin measurement in the food and from the bacteria could not be performed due to lack of appropriate methods. This could also be the reason why a lot of egc caused SFPOs remain undiscovered. In this study for two enterotoxins (SEG and SEI) out of the five egc enterotoxins, an ELISA method was available, whereas for the other egc enterotoxins this is still not the case. Seu was not considered at all as there is no literature demonstrating its emetic activity. Due to this lack of information about egc enterotoxins, new methods and tools need to be developed in order to better understand and predict their expression and regulation mechanisms [START_REF] Zeaki | The Role of Regulatory Mechanisms and Environmental Parameters in Staphylococcal Food Poisoning and Resulting Challenges to Risk Assessment[END_REF]. In consequence the aim of the present study was to determine whether WGS data can be used to predict staphylococcal enterotoxin production of the egc in vitro, particularly of SEG and SEI.

Prediction of SEG and SEI production in vitro

For the present study, 75 strains were chosen, originating from both human hosts and animal (cattle), as well as from environmental and food sources, with a special attention to SFPO strains [START_REF] Merda | NAuRA: Genomic Tool to Identify Staphylococcal Enterotoxins in Staphylococcus aureus Strains Responsible for FoodBorne Outbreaks[END_REF]. Out of the 75 strains, 60 were allocated to the 15 previously defined vSaβ types [START_REF] Klaui | Characterization and Comparative Analysis of the Staphylococcus aureus Genomic Island vSabeta: an In Silico Approach[END_REF]. The remaining 15 strains could be grouped into 7 newly defined vSaβ types (Figure 22). According to these new insights, using the vSaβ types seems to be a very precise tool to characterize the different egc present in S. aureus strains, instead of using egc types I to VI as it has been described previously [START_REF] Chieffi | Novel insights into the enterotoxigenic potential and genomic background of Staphylococcus aureus isolated from raw milk[END_REF][START_REF] Collery | Associations between enterotoxin gene cluster types egc1, egc2 and egc3, agr types, enterotoxin and enterotoxin-like gene profiles, and molecular typing characteristics of human nasal carriage and animal isolates of Staphylococcus aureus[END_REF][START_REF] Jarraud | egc, a highly prevalent operon of enterotoxin gene, forms a putative nursery of superantigens in Staphylococcus aureus[END_REF][START_REF] Letertre | Identification of a new putative enterotoxin SEU encoded by the egc cluster of Staphylococcus aureus[END_REF][START_REF] Thomas | Staphylococcal enterotoxin-like toxins U2 and V, two new staphylococcal superantigens arising from recombination within the enterotoxin gene cluster[END_REF].

The present study shows that there are two groups of SE producers, strains that produce low levels of SEG and SEI and strains with increased SE production (for both SE P < 0.001). A special case is the absence of SEG production for vSaβ IV and XXI. This is explained by the fact that both had a truncated seg gene, resulting in an incomplete, non-detectable protein.

A very high linear dependency was observed between the production of SEG and SEI (R = 0.98, P < 0.001) while the amount of SEI measured was approximately 16 times higher than the one of SEG. The high correlation between SEG and SEI production suggests that both SE are regulated primarily by the same transcription factor as proposed by Kusch et al. [START_REF] Kusch | The influence of SaeRS and sigma(B) on the expression of superantigens in different Staphylococcus aureus isolates[END_REF]. This hypothesis however neglects the fact that the SEG production is 16 x lower than for SEI accounting for a fine tuning by additional transcriptions factors as observed for other SE [START_REF] Fisher | Basis of Virulence in Enterotoxin-Mediated Staphylococcal Food Poisoning[END_REF][START_REF] Kusch | The influence of SaeRS and sigma(B) on the expression of superantigens in different Staphylococcus aureus isolates[END_REF].

During the first robust linear regression analysis outliers were observed (G68P, 18SBCL669 and 05CEB52). For these strains, all members of vSaβ types XI and XIX, production of SEI was always lower in relation to the SEG production (Appendix II, Figure S2). As demonstrated in Table 11, SEI of both vSaβ types show the lowest similarity (93%) when compared to the reference (Mu50). These findings may indicate that the monoclonal antibody used for the present study matches incompletely with the SEI epitopes produced by vSaβ type XI and XIX producing strains, resulting in a reduced detection of SEI quantities. Beside the technical aspect it cannot be ruled out, however that regulation of SEI production is special for these vSaβ types. To clarify this ambiguities, additional studies are required.

The results of the present European study were not in agreement with the results published from Omoe et al. [START_REF] Omoe | Detection of seg, seh, and sei genes in Staphylococcus aureus isolates and determination of the enterotoxin productivities of S. aureus isolates Harboring seg, seh, or sei genes[END_REF], who detected SEI only in 40% of the strains and SEG

was not detected at all. In our study SEG was produced by 96 % of the strains and SEI for 100% of the strains being positive for the two enterotoxins genes as detected by NAuRA [START_REF] Merda | NAuRA: Genomic Tool to Identify Staphylococcal Enterotoxins in Staphylococcus aureus Strains Responsible for FoodBorne Outbreaks[END_REF]. Only for one strain (18SBCL678), seg was predicted, but SEG enterotoxin was not detected. As our results were generated from a large variety of strains, the involvement of the egc enterotoxins in SFPO should therefore be reconsidered.

Inferring the origin of an SFPO-involved strain

Looking at the major vSaβ types found in this study (I, III, IV, XII, XXII) it was observed that in each group there are SFPO associated strains (isolated from food) and strains that are human-(infection) OR cattle-derived (mastitis), but never both for the same vSaβ type.

In addition to our previous study with 15 allocated vSaβ type observed [START_REF] Klaui | Characterization and Comparative Analysis of the Staphylococcus aureus Genomic Island vSabeta: an In Silico Approach[END_REF], we found 9 new types. Again, a perfect concordance between vSaβ type and CC was found, confirming this observation as a general principle in S. aureus. This principle can now be applied for evaluation of egc containing strains, involved in SFPO. In fact, instead of inferring the vSaβ type involved in the SFPO, the common and simpler method of CC assessment can be performed. This is particularly easy for WGS data, as the reads can be directly uploaded to some internet app such as cge.cbs.dtu.dk for inferring of the sequence type (ST) which is then used together with the pubMLST database program [START_REF] Jolley | Open-access bacterial population genomics: BIGSdb software, the PubMLST.org website and their applications[END_REF] to obtain the corresponding CC. If WGS data are not available, the standard MLST procedure can be performed using standard PCR and Sanger sequencing for the seven housekeeping genes (arcC, aroE, glpF, gmk, pta, tpiA, yqiL) [START_REF] Enright | Multilocus sequence typing for characterization of methicillin-resistant and methicillin-susceptible clones of Staphylococcus aureus[END_REF]. Instead of the original primers (35), the newly designed primer by Boss et al. [START_REF] Boss | Bovine Staphylococcus aureus: Subtyping, evolution, and zoonotic transfer[END_REF]) can be applied. They enable unidirectional Sanger sequencing, which considerably saves cost, work, and time.

The suspected reason for this strong link between CC and vSaβ type is that vSaβ acquisition by horizontal transfer in the S. aureus genome happened immediately before or simultaneously to clonal diversification of S. aureus [START_REF] Klaui | Characterization and Comparative Analysis of the Staphylococcus aureus Genomic Island vSabeta: an In Silico Approach[END_REF]. This hypothesis is also supported by the phylogenetic analysis of the core genomes of the present study (Figure 23), showing a perfect agreement between the phylogenetic clade, CC, and vSaβ type.

The CC can be used to perform an association of an egc carrying SFPO strain to a specific origin (human or cattle). As it can be seen in Figure 23, CC705 and CC20 are strains strictly associated with bovine mastitis and dairy products. In fact CC705 positive strains are classical pathogens of bovine mastitis observed in and outside Europe [START_REF] Boss | Bovine Staphylococcus aureus: Subtyping, evolution, and zoonotic transfer[END_REF]Cremonesi et al., 2015;[START_REF] Kappeli | Staphylococcus aureus related to bovine mastitis in Switzerland: Clonal diversity, virulence gene profiles, and antimicrobial resistance of isolates collected throughout 2017[END_REF]. In addition they are also frequently present in delivered milk [START_REF] Boss | Bovine Staphylococcus aureus: Subtyping, evolution, and zoonotic transfer[END_REF] and cheese (Hummerjohann et al., 2014). CC705 strains are uniquely positive for spa type t529 (Table 10) and are typical colonizers of bovine skin as well as infections of bovine wounds [START_REF] Leuenberger | Genotypes of Staphylococcus aureus: On-farm epidemiology and the consequences for prevention of intramammary infections[END_REF]. Similar findings are also true for CC20 positive strains. These can also cause bovine mastitis and are present in delivered milk, but they are less abundant than CC705 [START_REF] Boss | Bovine Staphylococcus aureus: Subtyping, evolution, and zoonotic transfer[END_REF].

On the other hand strains allocated to CC5, CC30, and CC45 were exclusively isolated from human samples (infection, skin, faeces, nose, and throat) and from food (Figure 23) where human handling was very likely (ready to eat products). Furthermore these CCs are widely described in literature to be found in human infections [START_REF] Donker | The population structure of Staphylococcus aureus among general practice patients from The Netherlands[END_REF][START_REF] Elie-Turenne | Prevalence and characteristics of Staphylococcus aureus colonization among healthcare professionals in an urban teaching hospital[END_REF][START_REF] Espadinha | Extensive dissemination of methicillin-resistant Staphylococcus aureus (MRSA) between the hospital and the community in a country with a high prevalence of nosocomial MRSA[END_REF][START_REF] Resman | The prevalence, population structure and screening test specificity of penicillin-susceptible Staphylococcus aureus bacteremia isolates in Malmo, Sweden[END_REF]. This is a further advantage of CC nomenclature as literature about them is broad [START_REF] Argudin | Genotypes, exotoxin gene content, and antimicrobial resistance of Staphylococcus aureus strains recovered from foods and food handlers[END_REF][START_REF] Baumgartner | Virulence and resistance gene profiles of staphylococcus aureus strains isolated from ready-to-eat foods[END_REF][START_REF] Suzuki | Molecular epidemiological characterization of Staphylococcus aureus isolates originating from food poisoning outbreaks that occurred in Tokyo, Japan[END_REF] enabling to extend the scope beyond an egc enterotoxin caused SFPO.

Application of the new insights in evaluation of egc-caused SFPOs

The involvement of egc enterotoxins in foodborne outbreaks is highlighted by the fact that vSaβ type (and the corresponding CC) from S. aureus strains producing high levels of SEG and SEI are also described to be involved in foodborne outbreaks, especially CC5, CC20, and CC45 [START_REF] Argudin | Genotypes, exotoxin gene content, and antimicrobial resistance of Staphylococcus aureus strains recovered from foods and food handlers[END_REF][START_REF] Suzuki | Molecular epidemiological characterization of Staphylococcus aureus isolates originating from food poisoning outbreaks that occurred in Tokyo, Japan[END_REF][START_REF] Umeda | Molecular and epidemiological characterization of staphylococcal foodborne outbreak of Staphylococcus aureus harboring seg, sei, sem, sen, seo, and selu genes without production of classical enterotoxins[END_REF]. Furthermore, certain strains of CC45 (harbouring egc) do not harbour any classical enterotoxin (Johler, Weder, et al., 2015;[START_REF] Umeda | Molecular and epidemiological characterization of staphylococcal foodborne outbreak of Staphylococcus aureus harboring seg, sei, sem, sen, seo, and selu genes without production of classical enterotoxins[END_REF], yet these strains could have been involved in foodborne outbreaks.

As an example we deal with strain 18SBCL673, that was involved in a foodborne outbreak related to artisanal goat cheese in southern Switzerland (Johler, Weder, et al., 2015) and was included in the present study. It is characterized by the presence of just egc enterotoxins, as shown by NAuRA, and produces a substantial amount of SEG (2.04 +/-0.33 ng/ml) and SEI (40.58 +/-9.03 ng/ml). It is positive to vSaβ type XXII and CC45. As the strain had been isolated from goat cheese, it could be hypothesized that goat milk was the probable source. However, according to the present study (Figure 23), it is clear that the origin of the involved strain is with a high probably human. As a consequence the SFPO caused by this strain was a highly human contamination during cheese manufacturing. This conclusion is supported by the fact that CC45 is never found in goats and goat milk [START_REF] Merz | Staphylococcus aureus Isolates from Goat and Sheep Milk Seem to Be Closely Related and Differ from Isolates Detected from Bovine Milk[END_REF][START_REF] Porrero | Clonal diversity of Staphylococcus aureus originating from the small ruminants goats and sheep[END_REF].

Conclusion

The presented study demonstrates that the in vitro production of SEG and SEI can be predicted based on the vSaβ-type and the CC of a strain. Furthermore, the vSaβ-type/CC enables to predict the source of an egc-positive SFPO strain (animal or human derived). Due to the perfect correlation between CC and vSaβ type, the use of common CC typing is an easy and quick way to characterize a strain involved in a SFPO. It is therefore a good alternative to the proposed egc typing (I-IV), method which results in only four biological irrelevant types.

This information will enhance the ability to better understand the involvement of the egc enterotoxins in SFPO. The fact that the egc is found in more than 50% of the S. aureus strains and, according to our study, exact 75% expressed SEG and 100% expressed SEI, is a further indication that these and another egc enterotoxins are involved in SFPO.

Materials and methods

Bacterial strain and genome collection

The general aim was to use egc harbouring S. aureus strains representing a large diversity in their genomes and origins. In order to achieve this, 75 strains and genomes from different sources (food, environment, animal, human), as well as different geographical origins were chosen (Table 10). SFPO genome sequences and strains (42 genomes and strains) were obtained from the collection of the European National Reference Laboratory for Coagulase

Positive Staphylococci (EURL CPS, Maisons-Alfort, France). Nine Swiss bovine mastitis strains were used from the Agroscope strain and genome collection, these strains have been sampled previously by Fournier et al. (Fournier et al., 2008) and their genome sequenced by [START_REF] Klaui | Characterization and Comparative Analysis of the Staphylococcus aureus Genomic Island vSabeta: an In Silico Approach[END_REF]. For genomic and phylogenetic analysis seven strains of human and animal origin were obtained from NCBI (reference sequence database, https://www.ncbi.nlm.nih.gov) in order to increase the sample size and variation of the strains. Two strains (Mu50 and N315)

were obtained from P. Moreillon (University of Lausanne). Thirteen egc containing strains were isolated from food in Switzerland (details see paragraph below). An overview of the bacterial strain collection used in study is listed in Table 10.

Characterization of Swiss food strains

Starting with 45 Swiss S. aureus strains originating from food were obtained from the Federal Food Safety and Veterinary Office (kindly provided by A. Baumgartner). The presence of egc genes in these strains was determined by applying a real-time PCR assay with melting curve analysis (mPCR) for detection of seg, sei, sem, sen, and seo. For detection of seg and sei primers and PCR conditions were applied as described by Cosandey et al. (Cosandey et al., 2016). For detection of sem, sen, and seo new primers were designed (Table 12). The detection of seu was omitted, as its emetic activity has not been shown so far. After being cultured at 37 °C for 24 h on blood agar (bioMérieux Suisse s.a., Geneva, Switzerland), DNA was extracted from single colonies of Staph. aureus. A colony was picked and resuspended in 100 µL of 10 mM Tris-HCl and 10 mM EDTA (pH = 8.5), incubated at 95°C for 10 min, and immediately stored on ice. The lysates were diluted 1:100 in H2O to be used as templates for the different mPCRs [START_REF] Boss | Bovine Staphylococcus aureus: Subtyping, evolution, and zoonotic transfer[END_REF]. For all mPCRs, the total volume was 20 µL containing 300 nM of corresponding forward and reverse primer (Table 12), 1× Kapa Sybr Fast (Kapa Biosystems Inc., Woburn, MA), and 2.5 µL of 1:100 diluted DNA template. The mPCR run began with an initial denaturation at 95 °C for 3 min, followed by 35 cycles of denaturation at 95 °C for 3 s, annealing and extension in a single step at 60 °C for 50 s, and a final extension step at 60 °C for 5 min. Melting of the amplicons was performed between 60 °C and 94 °C, with increments of 1 °C and a 5-s waiting time at each step. The mPCRs were performed using a Rotor-Gene 6000 real-time thermal cycler (Corbett Life Science, Mortlake, Australia). Primer specificity (Table 12) was tested with S. aureus strains that were previously sequenced namely G11F, G19P, M1280, M1655, M2323, M2682, M3783, Mu50, N315 (see Table 10 Applying the mPCR for detection of the egc genes showed that only 40% of the strains was egc-positive. Based on the diversity of their origins 14 egc-positive strains were selected.

These Swiss strains, isolated from food were sequenced as follows: strains were cultured at 37 °C for 24 h on blood agar, 3-4 colonies were suspended in 4.5 ml Tryptic Soy broth (TSB, Becton Dickinson, New Jersey, USA) and incubated 18 h (37 °C, with shaking). From this overnight culture (ONC) 1 ml was suspended in 500 ml TSB and incubated at the same conditions. The resulting ONC was centrifuged for 23 min (7 °C, 6'000 x g) (Cellsep 6/720R, Henderson Biomedical Ltd, Lower Sydenham, UK). The supernatant was discarded and the pellet resuspended in 15 ml 10 mM; Tris/HCl pH 7.8 and transferred to a falcon tube which again was centrifuged for 5 min (4 °C, 18'000 x g). After centrifugation the pellet was treated using the NucleoBond® Xtra Maxi Kit (Machery Nagel, Düren, Germany) according to the manufacturers protocol with following modifications: instead of resuspending the pellet directly in 24 ml RES (from the kit), the pellet was resuspended in 2 ml RES containing 350 mg glass beads (425-600 μm, Merck, Darmstadt, Germany ) was added and shaked on the Bead Ruptor at level 6 for 45 sec (Bead Ruptor Elite, Omni international, Kennesaw GA, USA). After centrifugation for 5 min (4 °C, 13'500 x g), 22 ml were added to the supernatant and DNA was then extracted according to the protocol of the manufacturer of the kit.

Bioinformatics

The reads from the strains from EURL CPS were obtained from the European Nucleotide Archive database (https://www.ebi.ac.uk/ena). For these reads and the reads from the Swiss food strains, the method for assembly and annotation was applied according to Merda et al. [START_REF] Merda | NAuRA: Genomic Tool to Identify Staphylococcal Enterotoxins in Staphylococcus aureus Strains Responsible for FoodBorne Outbreaks[END_REF]. Before the assembly, reads were normalized using BBnorm (https://jgi.doe.gov/data-and-tools/bbtools/) to have a maximum coverage of 100x. Normalized reads were trimmed using Trimmomatic [START_REF] Bolger | Trimmomatic: a flexible trimmer for Illumina sequence data[END_REF]. Then, quality filtering was performed removing reads shorter than 50 bp, as well as excluding bases having a Phred quality score lower than 30. With these filtered reads, assembly was performed in three steps:

(i) a de novo assembly was generated using SPAdes (v.3.9.1) [START_REF] Bankevich | SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing[END_REF] applying the default parameters, (ii) scaffolding was performed in MeDuSa [START_REF] Bosi | MeDuSa: a multi-draft based scaffolder[END_REF], using the nearest complete public genome of S. aureus estimated by Mash [START_REF] Ondov | Mash: fast genome and metagenome distance estimation using MinHash[END_REF], and (iii) gaps were closed using GMcloser [START_REF] Kosugi | GMcloser: closing gaps in assemblies accurately with a likelihood-based selection of contig or long-read alignments[END_REF]. The quality of each assembled genome was assessed with QUAST (v.4.3) [START_REF] Gurevich | QUAST: quality assessment tool for genome assemblies[END_REF]. The assemblies were annotated using Prokka (v.1.11) [START_REF] Seemann | Prokka: rapid prokaryotic genome annotation[END_REF] and RAST [START_REF] Aziz | The RAST Server: rapid annotations using subsystems technology[END_REF] for the prediction of coding sequences (CDSs).

MLST, spa type and vSaβ type allocation

For all 75 genomes used in this study, three typing methods were applied to further characterize the strains genomically: (i) Multilocus sequence typing (MLST), (ii) spa typing, and (iii) vSaβ typing. The MLST of the seven housekeeping genes [START_REF] Larsen | Multilocus sequence typing of total-genome-sequenced bacteria[END_REF] and spa typing [START_REF] Bartels | Comparing wholegenome sequencing with Sanger sequencing for spa typing of methicillin-resistant Staphylococcus aureus[END_REF] were done by using the Center for Genomic Epidemiology online platform (http://www.genomicepidemiology.org/). In the pubMLST database program [START_REF] Jolley | Open-access bacterial population genomics: BIGSdb software, the PubMLST.org website and their applications[END_REF], the sequence types (STs) from the MLST were used to allocate each strain to a clonal complex (CC), for ST504 in the actual pubMLST database no corresponding CC is available, in consequence, this ST was allocated to CC705 as also described in literature [START_REF] Boss | Bovine Staphylococcus aureus: Subtyping, evolution, and zoonotic transfer[END_REF]. vSaβ islands were identified in the genome by applying the method described by [START_REF] Klaui | Characterization and Comparative Analysis of the Staphylococcus aureus Genomic Island vSabeta: an In Silico Approach[END_REF]. Briefly, if the vSaβ island of a strain had a sequence similarity of ≥ 90 % to the reference strain of any existing vSaβ type, it was considered of the same type [START_REF] Klaui | Characterization and Comparative Analysis of the Staphylococcus aureus Genomic Island vSabeta: an In Silico Approach[END_REF]. If the sequence similarity was < 90 % the vSaβ island was defined as a new type. All alignments were performed by using the Needleman-Wunsch algorithm of Clone Manager Professional 9 software (Scientific & Educational Software, Denver, CO).

Enterotoxin genes profiles

The enterotoxin genes profiles of the S. aureus strains, based on the WGS, were determined using the NAuRA tool (https://github.com/afelten-Anses/NAuRA). The screening of the enterotoxins was performed using the gene sequence and their relative protein sequence of the already described 26 SE and the estimated parameters of Blast in Merda et al. (Merda et al., 2020).

Phylogenetic analysis

The core genome of each of the 75 strains was determined by Roary pipeline [START_REF] Page | Roary: rapid large-scale prokaryote pan genome analysis[END_REF]. For this, the previously obtained GFF3 file from Prokka was used as an input, containing all the strains' genes as detected by the software. All genes of a strains' core genome were then concatenated. Then a multiple sequence alignment (MSA) (using MAFFT (Katoh et al., 2002)) was performed using the concatenated core genomes of all the strains. The MSA was then imported into the Gblock program [START_REF] Castresana | Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis[END_REF] for quality checking using the default setup, and removing any misaligned regions. A phylogenetic tree was constructed using the maximum-likelihood method in IQtree [START_REF] Trifinopoulos | W-IQ-TREE: a fast online phylogenetic tool for maximum likelihood analysis[END_REF]. This program estimated the evolutionary model of sequences, and the best model, according Akaike criterion, was GTR + I + gamma. The branch support was calculated by the bootstrap method, using 1000 replicates. The graphic representation of the phylogeny was obtained by using iTOL web viewer (https://itol.embl.de/) (Letunic and Bork, 2019).

Staphylococcal enterotoxin measurement

The 32 S. aureus strains used for the enterotoxin measurements are shown in Figure 23 and Figure 24. These were selected based on their allocation to the different vSaβ islands (Table 10). If, available, three strains per vSaβ type were used. The selected strains were cultivated on plate count agar (PCA, Becton Dickinson, Franklin Lakes, USA) for 24 h at 37 °C, then 3 single colonies were taken and suspended in 45 ml brain heart infusion broth (BHI, Becton Dickinson). The inoculated broth was then incubated at 37 °C for 24 h on a flask and shaking.

After 24 h, optical density of the culture at was measured in order to check the growing of the cells (OD480 > 1.8). The culture was transferred to a falcon tube and centrifuged at 8'000 x g for 15 min at room temperature. The supernatant was then filtered through a 0.2 µm syringe filter and the resulting filtrate was then used for the downstream analysis. Quantitative analysis of SEG and SEI was performed by using an in-house sandwich quantitative ELISA. seg (Genbank CP001781.1) and sei (Genbank CP001781.1) genes from S. aureus were synthetized (Genecust) and inserted into a bacterial plasmid (IPTG inducible pET22b(+) vector Novagen, Merck) for inducible expression of recombinant SEG and SEI toxins (thereafter used as immunogens and standards). Specific lab-made monoclonal antibodies were used as coating and probing biotinylated antibodies. Briefly, Biozzi mice were immunized 4 times at 3 week-interval with 10 μg of recombinant SEG or SEI toxin in Alum adjuvant (intraperitoneal injection). After intravenous boost injections, hybridomas were produced by fusing spleen cells with NS1 myeloma cells, as previously described by Köhler and Milstein (Köhler, 1975).

Monoclonal antibodies were produced from hybridoma culture supernatants and further purified by protein A or protein G affinity chromatography using AKTAxpress system (GE Heathcare, Chicaco, USA).

Two separate 96-well polystyrene microtiter plates (MaxisorpTM, Nunc, Roskilde, Denmark)

were coated with 100 µL of monoclonal anti-SEG IgG or anti-SEI IgG (SEG41 and SEI27) at 10 µg/mL in PBS 50 mM pH7.4 overnight at room temperature (RT), blocked with 300 µL/well of buffer Enzyme Immuno Assay (PBS 0.1 M pH 7.4, Bovine Serum Albumine 1 g/L, Sodium Azide 0.1 g/L) for at least 4 hours at RT. Saturated microplates were washed by 300 µL of phosphate/tween20 before use. A calibration curve was prepared with dilutions of SEG and SEI purified recombinant toxins with five concentration levels between 0 -0.3 ng SEG/mL and 0 -2.0 ng SEI/mL, respectively (duplicate calibration points per level). 100 µL/well of samples and recombinant standard toxins were distributed and incubated at RT for 60 min, washed three times with PBS/Tween 20, followed by addition of 100 ng/mL of biotinylated monoclonal anti-SEG or anti-SEI antibody (SEG27 and SEI26) at RT for 60 min. After extensive washing, 100 µL/well of poly-horseradish peroxidase-labelled streptavidin (dilution 1/50 000, Thermo

Fisher Scientific) was used for detection at RT for 30 min, and washed 5 times again. Then, 100 µL/well of substrate solution containing tetramethylbenzidine (TMB, Thermo Fisher Scientific, Waltham, USA) was added for 30 min. Finally, the reaction was stopped by 100 µL of H2SO4 2 N. Absorbances were read at 450 nm in a microplate reader (SAFAS, Monaco).

Quantification was performed by using a calibration curve based on the quadratic fit model.

Validation data (sensitivity, specificity and repeatability) of the above described method are published in [START_REF] Féraudet Tarisse | Highly Sensitive and Specific Detection of Staphylococcal Enterotoxins SEA, SEG, SEH, and SEI by Immunoassay[END_REF].

Statistical analysis

For analysis of potential correlation between production of SEG and SEI a regression analysis was performed. First, the robust method was applied to verify the regression model and to identify outliers. Four outliers were identified and eliminated from the dataset before calculating an ordinary least square regression model.

To proof the two different levels of SEG and SEI production a Kruskal-Wallis test was performed. For all statistical analysis measured values under the limit of detection were taken with the value 0. All statistical analyses were performed in Systat (Version 13, Systat, Chicago, USA).

The graphical presentation of the enterotoxin data was performed using R (Version 3.4.4) with the packages ggplot [START_REF] Wickham | ggplot2: Elegant Graphics for Data Analysis[END_REF], ggsignif (Ahlmann-Eltze, 2019), and ggpubr [START_REF] Kassambara | MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform[END_REF]. With these packages the data of the enterotoxin production of the single strains were plotted in increasing order of production (mean +/-standard deviation) and a colour given according to their relative vSaβ type.

Résumé (French)

Selon l'Autorité européenne de sécurité des aliments ( 2019 L'étude donne un aperçu de la production d'entérotoxines egc dans des conditions de stress.

Ces informations permettront d'améliorer la disponibilité des méthodes de contrôle des entérotoxines egc dans la production et le stockage des aliments.

Introduction

Staphylococcal food poisoning (SFP) is an intoxication caused by ingestion of food containing one or more enterotoxin produced by Staphylococcus aureus (S. aureus). The symptoms of SFP include nausea and violent vomiting, abdominal cramps and diarrhea appearing 2-8 hours after ingestion of contaminated food. SFP is often self-limiting with recovery occurring 1-3 days after the onset of symptoms (Le [START_REF] Loir | Staphylococcus and food poisoning[END_REF]. However, symptoms may be more severe for the young, elderly and immunocompromised [START_REF] Argudin | Food poisoning and Staphylococcus aureus enterotoxins[END_REF][START_REF] Murray | Recognition and management of Staphylococcus aureus toxin-mediated disease[END_REF]. Because symptoms often disappear in a short time, most of the SFP remain undiscovered as people don't need to get hospitalized and in consequence many cases go unreported. Nevertheless, its frequency has a significant impact on the economy, resulting in a loss in productivity [START_REF] Fisher | Basis of Virulence in Enterotoxin-Mediated Staphylococcal Food Poisoning[END_REF]. It also represents a serious financial burden, especially for the food industry, catering businesses, and public healthcare systems [START_REF] Fisher | Basis of Virulence in Enterotoxin-Mediated Staphylococcal Food Poisoning[END_REF] Staphylococcal enterotoxin (SE) are stable to heat, digestive proteinases, irradiation, denaturing agents, and a wide pH range [START_REF] Tatini | Influence of food environments on growth of Staphylococcus aureus and production of various enterotoxins[END_REF]. Especially protein rich foods as dairy products were reported to be more protective to SEs [START_REF] Fung | Thermal inactivation of staphylococcal enterotoxin B and C[END_REF]Hennekinne et al., 2012;Tatini et al., 1971). SEs have the ability to traverse the harsh acidic conditions within the stomach to reach the intestine, and can cause SFP in the absence of live bacteria. Typically, only high nanogram to low microgram quantities of enterotoxins are needed to induce the symptoms of SFP [START_REF] Hu | Induction of emetic response to staphylococcal enterotoxins in the house musk shrew (Suncus murinus)[END_REF][START_REF] Larkin | Staphylococcus aureus: the toxic presence of a pathogen extraordinaire[END_REF][START_REF] Ono | The emetic activity of staphylococcal enterotoxins, SEK, SEL, SEM, SEN and SEO in a small emetic animal model, the house musk shrew[END_REF][START_REF] Ono | Identification and characterization of two novel staphylococcal enterotoxins, types S and T[END_REF]. An additional characteristics is that a great diversity of staphylococcal enterotoxins exists. Up to date 26 enterotoxins have been described in literature [START_REF] Benkerroum | Staphylococcal enterotoxins and enterotoxin-like toxins with special reference to dairy products: An overview[END_REF]. However, only the classical enterotoxins (SEA-SEE) can be detected by commercially kits [START_REF] Hennekinne | Intralaboratory validation according to the EN ISO 16 140 Standard of the Vidas SET2 detection kit for use in official controls of staphylococcal enterotoxins in milk products[END_REF]. Literature is showing that also the other (non-classical) enterotoxins seem to be involved in staphylococcal food poisoning outbreak (SFPO) [START_REF] Ciupescu | Characterization of Staphylococcus aureus strains and evidence for the involvement of non-classical enterotoxin genes in food poisoning outbreaks[END_REF][START_REF] Ikeda | Mass outbreak of food poisoning disease caused by small amounts of staphylococcal enterotoxins A and H[END_REF][START_REF] Jorgensen | An outbreak of staphylococcal food poisoning caused by enterotoxin H in mashed potato made with raw milk[END_REF]. Out of them, the enterotoxin gene cluster (egc) enterotoxins have been described to be involved in outbreaks. One was caused by goat cheese in Switzerland [START_REF] Johler | Further evidence for staphylococcal food poisoning outbreaks caused by egc-encoded enterotoxins[END_REF] and another one with ready to eat food in Japan [START_REF] Umeda | Molecular and epidemiological characterization of staphylococcal foodborne outbreak of Staphylococcus aureus harboring seg, sei, sem, sen, seo, and selu genes without production of classical enterotoxins[END_REF] . The egc enterotoxins are six successive enterotoxin genes (seg, sei, sem, sen, seo, seu) located on the vSaβ, which has been described to be a phage that is often incomplete in S. aureus nowadays [START_REF] Klaui | Characterization and Comparative Analysis of the Staphylococcus aureus Genomic Island vSabeta: an In Silico Approach[END_REF]. Out of these six enterotoxins, emetic activity has been described for 5 out of them [START_REF] Ono | The emetic activity of staphylococcal enterotoxins, SEK, SEL, SEM, SEN and SEO in a small emetic animal model, the house musk shrew[END_REF]. In addition literature describes the presence of the egc in almost 50% of the S. aureus strains that are omnipresent in the different sources of isolation (human, animal, environment, food) [START_REF] Argudin | Food poisoning and Staphylococcus aureus enterotoxins[END_REF][START_REF] Smyth | Superantigen genes encoded by the egc cluster and SaPIbov are predominant among Staphylococcus aureus isolates from cows, goats, sheep, rabbits and poultry[END_REF]. These facts show the need to further characterize the behaviour of egc enterotoxins especially with respect to the food production conditions under which these enterotoxins are secreted. From S. aureus it is known that it is able to grow under a wide range of temperature, pH, and NaCl concentrations and thus the types of foods where S. aureus can be present vary from ready-to-eat meals to fermented foods [START_REF] Adams | Food Microbiology[END_REF]Le Loir et al., 2003). Regarding the condition of expression of egc enterotoxin, only few information are available. A study by [START_REF] Derzelle | Differential temporal expression of the staphylococcal enterotoxins genes during cell growth[END_REF] showed that egc enterotoxin seem to be expressed during the exponential growing phase. Another study investigated the impact of different food preservative like sodium nitrite, chitosan and Tea cathechin on the production of SEI (Zhao et al., 2017a). No information is available about the influence of classical food parameters like pH, temperature and NaCl on the production of none of the egc enterotoxins. In literature it has been described that phage borne se-genes are activated by phage inducing agents like low pH, oxidative stress, mitomycin C, NaCl and acetic acid [START_REF] Cao | Elevated enterotoxin A expression and formation in Staphylococcus aureus and its association with prophage induction[END_REF]Sumby and Waldor, 2003a;[START_REF] Wallin-Carlquist | Acetic acid increases the phage-encoded enterotoxin A expression in Staphylococcus aureus[END_REF][START_REF] Zeaki | Assessment of high and low enterotoxin A producing Staphylococcus aureus strains on pork sausage[END_REF].

The aim of the present study was to characterize the expression of egc enterotoxins during cell growth and to improve the understanding about the impact of heat and salt concentration on the egc enterotoxin production. This information will help to minimize their expression during food production and storage.

Material and Methods

Bacterial strains and cultivation conditions

Bacterial strains used in this study are listed in Table 13. These eight strain are a subset of 32 S. aureus strains that were previously characterized and their potential enterotoxin production (SEG and SEI) measured [START_REF] Schwendimann | Staphylococcal enterotoxin gene cluster: prediction of enterotoxin (SEG and SEI) production and of the source of food poisoning based on vSabeta typing[END_REF]. For this study the selection of eight was performed based on their genomic diversity, their behaviour in enterotoxin G and I production and their involvement in foodborne outbreaks [START_REF] Schwendimann | Staphylococcal enterotoxin gene cluster: prediction of enterotoxin (SEG and SEI) production and of the source of food poisoning based on vSabeta typing[END_REF].

Considering the different RNA expression, three strains out of these eight were chosen for a characterization of their behaviour under stress parameters. Prior to inoculation strains were grown on blood agar plates (BioMérieux Suisse s.a., Geneva, Switzerland) overnight (16 to 18 h) at 37 °C. A single colony was selected and transferred to 5 ml fresh BHI broth for pre-culture preparation (BHI, Becton Dickinson, Franklin Lakes, USA).

500 µl of the pre-culture was then used to inoculate 45 ml of BHI in a 100 mL bottle with screw top, according to the experimental set up.

Experimental set up and sampling

Experimental set up was performed according to Figure 25. 500 µL of pre-culture of each strain were inoculated in 45 ml BHI. 4 different kind of BHI with different concentration of NaCl were used: 5 g/L NaCl (standard), 12.5 g/L (adding 7.5 g/L NaCl), 20 g/L (adding 7.5 g/L NaCl) and

100 g/L NaCl (adding 95 g/L NaCl). All of them were incubated at standard conditions (37°C, 200 rpm). In addition standard BHI was also incubated at 45 °C and 200 rpm. Each single experiment was done in triplicate. Sampling points (T0-T3) were defined according to the behaviour of the single strains in the growing curve (Figure 26). T0 was immediately after inoculation, T1 in the mid-exponential phase, T2 at end of the exponential phase and T3 after 24 h. In order to check the growth of the bacteria OD480 was measured at each sampling point and crosschecked with the available growing curve of the respective strain. At each sampling point (T0, T1, T2) two samples (1 mL) were taken for expression analysis.

To each sample 800 µL of TEL (10 mM Tris-HCl and 10 mM EDTA, pH = 8.5) and 100 µL 10 10 CFU/ml L. casei were added and cells harvested by centrifugation (1 min at 4°C at 18'000 x g). Supernatant was discarded and the pellets resuspended in 400 µL TEL and 350 mg glass beads (425-600 μm, Merck, Darmstadt, Germany). Samples were then put on the Beat Ruptor Elite (Omni International, Kennesaw GA, USA) step 6 for 45 sec. After this procedure samples were put immediately on ice for 1 min and then centrifuged again (1 min at 4°C at 18'000 x g).

The supernatant of each samples was then divided into 2 separate tubes containing 100 µL each (one for RNA and one for DNA analysis). All samples were then immediately frozen in liquid nitrogen and stored at -80 °C until further analysis.

At each sampling point for ELISA (T1, T2, T3) 5 mL BHI were taken. The culture was transferred to a falcon tube and centrifuged at 8'000 x g for 15 min at room temperature. The supernatant was then filtered through a 0.2 μm syringe filter and the resulting filtrate was then used for the downstream analysis

RNA extraction and reverse transcription

Total RNA was isolated using the RNeasy plus mini kit (Qiagen, Hombrechtikon, Switzerland)

following the manufacturer's instructions. For this analysis 100 µL of the sample stored at -80°C (descriebed in experimental set up and sampling) adding 500 µl RTL plus was used. Two DNA contamination removal steps were included by passing the cell lysate through a genomic column and performing an on-column DNase digestion (RNase-Free DNase Set, Qiagen) of the samples bunt to the RNA spin column, as described by [START_REF] Sihto | Validation of reference genes for normalization of qPCR mRNA expression levels in Staphylococcus aureus exposed to osmotic and lactic acid stress conditions encountered during food production and preservation[END_REF]. The RNA templates were eluted in 50 µL RNase-free water and frozen immediately in liquid nitrogen.

Quantity and quality of RNA were assessed using QuickDrop spectrophotometer (Molecular Devices, San Jose, USA), RNA quality was considered sufficient if OD260/OD280 ≥ 1.8. To asses RNA integrity, the RNA Integrity Number (RIN) was measured using the miniaturized electrophoresis system RNA 6000 Pico Kit (Agilent Technologies, Santa Clara, CA, USA).

Samples were considered with RIN ≥ 7.0.

Reverse transcription was performed using the Quantitect Reverse Transcription Kit (Qiagen), using the optimized mix of oligo-dT provided in the kit. For each sample 480 ng of total RNA was converted to cDNA in a total volume of 20 µL. RT minus of each sample was also performed in order to be able to check if residual DNA was contained in the sample [START_REF] Sihto | Validation of reference genes for normalization of qPCR mRNA expression levels in Staphylococcus aureus exposed to osmotic and lactic acid stress conditions encountered during food production and preservation[END_REF].

DNA extraction

DNA extraction was performed using the High Pure PCR Template Preparation Kit, (Roche, Basel, Switzerland) following the manufacturer's instructions, taking the 100 µL sample stored at -80°C (descriebed in experimental set up and sampling) and adding 100 µL ddH2O (double distilled water) to the sample. In addition, elution was performed with 100 µL elution buffer instead of 200 µL and an additional centrifugation step was performed (10 min, 20'000 g).

Sample were then stored at -20°C until further analysis.

Quantitative real-time PCR (qPCR)

Each sample of cDNA was used for detection of the expressed quantities of seg, sei, sem, sen and seo. After reverse transcription, samples were diluted 1:10 using ddH2O. For qPCRs for detection of seg, sei, sem and sen, the total volume was 20 µL containing 300 nM of corresponding forward and reverse primer (Table 14), 1× Kapa Sybr Fast (Kapa Biosystems Inc., Woburn, MA, USA), and 2.5 µL of 1:10 diluted cDNA template. The qPCR for detection of seo used a concentration of 600 nM, instead of 300 nM, all other parameters were the same for all tested enterotoxin. The qPCR run starts with an initial denaturation at 95 °C for 3 min, followed by 35 cycles of denaturation at 95 °C for 10 s, annealing 57 °C for 50 s, extension at 72 °C for 10 s, and a final extension step at 60 °C for 210 s. Melting of the amplicons was performed between 60 °C and 94 °C, with increments of 1 °C and a 5-s waiting time at each step.

Each sample of isolated DNA was used to measure the exact amount of nuc copies (equal to number of cell). In order to do so the developed method of [START_REF] Graber | Development of a highly sensitive and specific assay to detect Staphylococcus aureus in bovine mastitic milk[END_REF][START_REF] Graber | Development of a highly sensitive and specific assay to detect Staphylococcus aureus in bovine mastitic milk[END_REF] was used with minor modifications. Primers and probes were used as presented in Table 14, a minor groove binder qPCR probe labelled with FAM at the 5′ end and with a confidential non fluorescent quencher at the 3′ end was used. Each 25 µL of qPCR mixture contained 1× Roche Light Cycler 480 Master Mix (Roche), 600 nM sense and antisense primers, 100 nM probe and 3.5 µL of extracted DNA (diluted 1:100 with ddH2O). The qPCR conditions were 95°C for 10 min followed by 45 cycles of 95°C for 15 s and 60° for 1 min.

The qPCRs were performed using a Rotor-Gene 6000 real-time thermal cycler (Corbett Life Science, Mortlake, Australia). Single peaks of the melting curve analysis confirmed the specificity of the amplicon. For each sample two separate qPCR reactions were performed

Standard curves

Standard curves based on genomic DNA were created by using amplicons of Staph. aureus genes seg, sei, sem, sen, seo and nuc. They were prepared by standard PCR (Table 14) using strains positive for these particular genes as described by [START_REF] Boss | Mastitis diagnostics: quantitative PCR for Staphylococcus aureus genotype B in bulk tank milk[END_REF][START_REF] Graber | Development of a highly sensitive and specific assay to detect Staphylococcus aureus in bovine mastitic milk[END_REF][START_REF] Sartori | Development of a new real-time quantitative PCR assay for the detection of Staphylococcus aureus genotype B in cow milk, targeting the new gene adlb[END_REF]. Shortly, standard PCR was performed in 25 μL containing 1× HotStarTaq Master Mix (Qiagen), 300 nM of each primer, and 2.5 μL of template. All corresponding primers are listed in Table 14. Standard PCR was performed using a Biometra cycler system (Biometra GmbH, Göttingen, Germany). The cycling conditions of the PCR were an initial denaturation step (95°C, 15 min), 35 cycles comprising denaturation at 94°C, annealing at 62°C and elongation at 72°C, each or 1 min, followed by a final elongation step at 72°C for 10 min and cooling to 4°C. The PCR products were then analyzed on the miniaturized electrophoresis system Agilent DNA 7500 Chip (Agilent Technologies, Basel, Switzerland) to test for specific PCR amplification. The products were purified with the Qiaquick PCR Purification Kit (Qiagen). Thereafter, the corresponding DNA concentrations were determined by spectroscopy (A260/A280, QuickDrop). Samples were diluted from 1 x 10 8 to 1 x 10 3 copies/2.5 µl.

Quantification of expression

In order to quantify the amount of expressed enterotoxin gene, the absolute quantification method, using an external calibration curve, was applied [START_REF] Pfaffl | Nucleic Acids -RNA Identification and Quantification via RT-qPCR[END_REF]. Expression is given in mRNA copies/cell, as values were normalized against the total amount of RNA and relativized to the amount of nuc copies which are equal to number of S. aureus cells (Figure 28A). In detail for quantification, the following procedure was applied: the mean value (n=2) coming from the standard curve for each enterotoxin gene (input Cp-values) was adjusted to the used volume before starting reverse transcriptase. This value was then normalized against the total RNA concentration [START_REF] Pfaffl | Nucleic Acids -RNA Identification and Quantification via RT-qPCR[END_REF] and relativized to the amount of nuc copies (DNA). The amount of nuc copies were also calculated based on the standard curve and 1 copy of nuc directly correlated with the number of cells (1 Staph. aureus cell contains exactly 1 nuc gene) [START_REF] Graber | Development of a highly sensitive and specific assay to detect Staphylococcus aureus in bovine mastitic milk[END_REF]. As a result the data are presented in expressed mRNA copies/ cell.

ELISA

Enterotoxins G and I were measured applying the method described by [START_REF] Féraudet Tarisse | Highly Sensitive and Specific Detection of Staphylococcal Enterotoxins SEA, SEG, SEH, and SEI by Immunoassay[END_REF] and further used in [START_REF] Schwendimann | Staphylococcal enterotoxin gene cluster: prediction of enterotoxin (SEG and SEI) production and of the source of food poisoning based on vSabeta typing[END_REF]. Results are presented in average +/-standard deviation.

Results

Growing curves

OD600 values measured during cell growth (standard conditions) showed no differences for the different strains used with the exception of Mu50 (Figure 26). Whereas for the other seven strains the mid-log was reached 2 hours after incubation, Mu50 started the log phase later, reaching the mid-log phase after 3 hours and 30 min. However, the late exponential phase was reached simultaneously by all strains (8 h 30 min after inoculation). When varying the sodium chloride concentration, differences could be observed (Figure 27). By applying 12.5 g/L or 20 g/L NaCl no differences in cell growth were observed comparing to 5 g/L NaCl with exception of Mu50 that started growing faster but reached the mid-log phase at the same time as the other strains did. On the other side by applying 100 g/L NaCl differences could be observed as mid-log phase was delayed and was observed only 4 h after inoculation for KS24 and 15SB and 6 h for Mu50. KS24 and 15SB could not reach the same optical density (between 1.6 and 1.8) as the other did (2.0). By applying an incubation temperature of 45 °C (Figure 27), differences between the strains could be observed. All strains started growing with the same speed like at 37°C, but the single strains were not able to keep the log phase for the same amount of time, meaning KS24 (after 5h) reached the end-log phase later than 15SB (2 h) and Mu50 (3 h 30 min). Based on the behavior of the single strains, sampling points (T1-T3) were adapted in order that sampling at mid-log and end-log phases could be adhered.

Expression and production of egc enterotoxin during the growing phase

Expression of egc enterotoxins during the growing phase was measured for eight strains at three different points in time: Immediately after inoculation (T0), at the mid exponential phase of cell growing (T1) and at the end of the log-phase (T2) . Expression is given in RNA copies/cell of S. aureus (Figure 28).

Under standard conditions, transcription of all se genes were successfully detected in all growth phases (Figure 28A). Although the relative abundance of each transcript differentially varied during growth, it appeared that each strain had a specific behaviour and its own pattern of expression, showing however also a general trend. The amount of mRNAs from the egc enterotoxins (seg, sei, sem, sen, and seo) tended to increase at the mid exponential growing phase and decreased shortly after, for some strains also the expression at T3 was measured, confirming again the further trend to decrease (data not shown).

Overall it can be observed that sem was the most expressed enterotoxin (maximum value T2, 13ceb, 2.34 * 10 2 +/-58.40 mRNA/cell, Figure 28A), wherever seo seem to be the fewest expressed (maximum value, T2, 13ceb, 4.99 * 10 1 +/-33.33 mRNA/cell). Seg seems also to be less expressed (maximum value, T2, 13ceb, 9.2 * 10 1 +/-27.30 mRNA/cell) than sei (maximum value, T2, 13ceb, 1.32 * 10 2 +/-40.10 mRNA/cell), this trend is also observed in the production of enterotoxin (Figure 28B) where concentration of SEI (T3, 13ceb, 109.40 +/-4.17 ng/mL) were always higher than the ones of SEG (T3, 13ceb 6.20 +/-0.09 ng/mL).

Strain 13ceb was overall the strain showing the highest mRNA expression (over all egc enterotoxin genes) and also the highest SEG and SEI production. On the other side for strains AG and 26.1 seg was not expressed at any point of time and repetition and also no enterotoxin could be measured for these strains.

By looking at the data of enterotoxin production (Figure 28B) measured at T1 to T3 it can be observed that production of SEG and SEI already started at T1, i.e. at the mid-exponential phase, just 2 h after inoculation showing values up 3.41+/-0.24 ng/mL for SEG and 64.57 +/-5.15 ng/mL for SEI. Production then increased again between T1 and T2 (end of exponential phase) showing values of 7.00 +/-0.22 ng/mL for SEG and 106.57 +/-2.38 ng/mL for SEI (13ceb). Between T2 and T3 (after 24 h) no additional production could be observed (T3 13ceb 6.20 +/-0.09 ng/mL SEG, 109.40 +/-4.17 ng/mL SEI).

Effect of NaCl concentration on the expression and production of egc enterotoxins

For the analysis of the effect of different NaCl concentrations on the expression and production of enterotoxin they were measured for three strains (Mu50, 15SB, KS24) with following NaCl concentrations: 5 g/L, 12.5 g/L, 20 g/L and 100 g/L (Figure 29). The three strains were selected based on the greatest diversity (genetic, expression behaviour, enterotoxin production).

Strains with very low or no production of enterotoxin were excluded, as they were considered not relevant. For all strains and conditions expression measurement showed a maximum at the mid exponential phase (T1) (see data in supplementary material). For the SEG and SEI measurements (Figure 29B) the highest concentration were found after 24 h (T3). In A B consequence in Figure 29 only data at the maximal concentration (T1 for mRNA and T3 for SEG and SEI) are shown. Overall NaCl seems to have an impact on the expression of the different enterotoxins at mRNA level and at protein level, even though enterotoxins could still be measured with concentrations of 100 g/L NaCl in BHI (T3, K24, 0.50 +/-0.13 ng/mL SEG, 10.54 +/-2.45 ng/mL SEI). The three strains, besides already producing different amounts of enterotoxins at standard conditions, also behave differently when adding different amounts of NaCl (100 g/L NaCl, KS24 10.54 +/-0.50 ng/mL SEI, Mu50 6.07 +/-0.79 ng/mL SEI, 15SB 11.730+/-4.25 ng/mL SEI).

Over all egc enterotoxins strain 15SB is having the highest expression with 12.5 g/L NaCl (1.62 * 10 2 +/-47.22 sem mRNA/cell) and decreasing for the higher NaCl concentrations (20 g/L NaCl, 7.05 * 10 1 +/-37.56 sem mRNA/cell ). For this strain this behaviour is only observable in transcription but not in enterotoxin production (12.5 g/L NaCl 37.63 +/-1.49 ng/mL SEI, 20 g/L NaCl 34.79 +/-5.87 ng/mL SEI). For strain KS24 for enterotoxins seg, sei, sem and seo after a reduction between 5 g/L and 12.5 g/L NaCl, expression seem to increase at 20 g/L NaCl and again decrease at 100 g/L. The same behaviour can also be observed at a protein level by looking at the production of SEG (2.70 +/-0.46 ng/mL with 12.5 g/L NaCl, 3.02 +/-0.31 ng/mL with 20 g/L NaCl) and SEI (30.60 +/-1.26 ng/mL with 12.5 g/L NaCl, 35.28 +/-0.23 ng/mL with 20 g/L NaCl) at the same NaCl concentrations.

Mu50 is constantly having a low expression (5 g/L NaCl, 6.39 * 10 0 +/-3.8 sei mRNA/cell) with no differences until 20 g/L (3.04 * 10 0 +/-3.37 sei mRNA/cell). Mu50 expression is continuously decreasing with increasing concentration of NaCl, the same behaviour can also be observed for SEG and SEI production (SEI 5 g/L NaCl 54.35 +/-8.99 ng/mL, 100 g/L NaCl 6.07 +/-0.79 ng/mL).

Under these conditions results show the maximum expression at the mid exponential phase (T1) (see data in supplementary material). For the SEG and SEI measurements, the highest concentration were found after 24 h (T3). Again just the data of T1 (mRNA) and T3 (enterotoxin G and I) are presented in Figure 30. Overall temperature seem to have an impact on the expression of the different enterotoxin at mRNA level and at a protein level, even though enterotoxin could still be measured (Mu50, T3, 15.13 +/-3.3 SEI) by an incubation at 45°C. The three strains, besides already producing different amounts of enterotoxins at standard conditions (37 °C), also behave differently when incubated at 45 °C (differences in enterotoxin between 37 and 45 °C, Mu50 reduction from 54.35 +/-8.99 to 15.13 +/-3.33 ng/mL SEI, 15SB reduction from 55.17 +/-1.20 to 6.44 +/-1.49 ng/mL SEI).

Over the three strains tested and enterotoxin types with a temperature of 45 °C a reduction in expression and enterotoxin production could be observed, with exception of Mu50 for genes sem and seo, which had a higher expression at T2 at 45°C (2.56 * 10 1 +/-24.18 sem mRNA/cell, 6.8 * 10 -1 +/ -0.55 seo mRNA/cell) rather than at 37 °C (1.50 * 10 1 +/-4.36 sem mRNA/cell, 4.6 * 10 -1 +/ -0.28 seo mRNA/cell). Overall it is remarkably that Mu50 produced 15.13 +/-3.33 ng/mL SEI at an incubation temperature of 45 °C after 24 h.

Discussion

In the present study we demonstrate the ability of the egc enterotoxin to be expressed and produced under food production like conditions. In fact, according to our knowledge no study has yet been published showing the effect of NaCl and temperature on the expression and production of egc enterotoxins. The findings of this study show that egc enterotoxins can be produced at NaCl concentration (12.5 and 20 g/L) that are normally applied in foods and are used as one component of the hurdle principle in food processing and preservation.

Previous studies were performed in order to study enterotoxin production under different stress parameters, but none was performed on the egc enterotoxins. The involvement of egc enterotoxins in foodborne outbreaks has been described largely in literature (Baumgartner et al., 2014;[START_REF] Johler | Further evidence for staphylococcal food poisoning outbreaks caused by egc-encoded enterotoxins[END_REF][START_REF] Umeda | Molecular and epidemiological characterization of staphylococcal foodborne outbreak of Staphylococcus aureus harboring seg, sei, sem, sen, seo, and selu genes without production of classical enterotoxins[END_REF][START_REF] Yan | Characterization of Staphylococcus aureus strains associated with food poisoning in Shenzhen, China[END_REF], even though in none of them strong evidence could be confirmed due to the lack of appropriate methods. In addition it has also been reported that more than 50% of the S. aureus strains may content these enterotoxins [START_REF] Borst | Phage-associated differences in staphylococcal enterotoxin A gene (sea) expression correlate with sea allele class[END_REF][START_REF] Song | Molecular Typing and Virulence Gene Profiles of Enterotoxin Gene Cluster (egc)-Positive Staphylococcus aureus Isolates Obtained from Various Food and Clinical Specimens[END_REF], what may lead to the conclusion that egc caused enterotoxin remain mostly undiscovered and are underestimated. Recently new methods were published being able to detect egc enterotoxins [START_REF] Féraudet Tarisse | Highly Sensitive and Specific Detection of Staphylococcal Enterotoxins SEA, SEG, SEH, and SEI by Immunoassay[END_REF][START_REF] Schwendimann | Staphylococcal enterotoxin gene cluster: prediction of enterotoxin (SEG and SEI) production and of the source of food poisoning based on vSabeta typing[END_REF]Zhao et al., 2017a;Zhao et al., 2017b), giving new insight into the production of these enterotoxins under standard conditions. As the egc enterotoxins seem to be involved in foodborne outbreaks it is however important to know under which conditions and parameters they are produced and their production could eventually be reduced during food production and storage. In consequence, the aim of the present study was to analyse the influence of NaCl and temperature on the expression of egc enterotoxins.

As an ELISA method is just available for detection of SEG and SEI, also a method based on RT-qPCR measuring the expression of enterotoxins seg, sei, sem, sen and seo was developed. For selu no method was developed as emetic activity for this enterotoxin has not already been demonstrated [START_REF] Hu | Induction of emetic response to staphylococcal enterotoxins in the house musk shrew (Suncus murinus)[END_REF][START_REF] Ono | The emetic activity of staphylococcal enterotoxins, SEK, SEL, SEM, SEN and SEO in a small emetic animal model, the house musk shrew[END_REF] . The strains used in this study were selected based on their difference in genetic background (different vSaβ and clonal complex) as well as their involvement in foodborne outbreaks [START_REF] Baumgartner | Virulence and resistance gene profiles of staphylococcus aureus strains isolated from ready-to-eat foods[END_REF][START_REF] Johler | Further evidence for staphylococcal food poisoning outbreaks caused by egc-encoded enterotoxins[END_REF][START_REF] Klaui | Characterization and Comparative Analysis of the Staphylococcus aureus Genomic Island vSabeta: an In Silico Approach[END_REF]. They were selected out of the 75 strains sequenced and further studied in [START_REF] Schwendimann | Staphylococcal enterotoxin gene cluster: prediction of enterotoxin (SEG and SEI) production and of the source of food poisoning based on vSabeta typing[END_REF]. The three strains selected for the different NaCl concentration and incubation temperature were selected based on the greatest diversity (genetic, expression behaviour, enterotoxin production). Strains with very low or no production of enterotoxin were excluded, as they were considered not relevant.

The developed RT-qPCR method was respecting the MIQE guidelines and their application in RT [START_REF] Taylor | A practical approach to RT-qPCR-Publishing data that conform to the MIQE guidelines[END_REF][START_REF] Taylor | The state of RT-quantitative PCR: firsthand observations of implementation of minimum information for the publication of quantitative real-time PCR experiments (MIQE)[END_REF]. In the past already different studies applied this method in order to study the ability to express enterotoxins in S. aureus [START_REF] Fleurot | Following pathogen development and gene expression in a food ecosystem: the case of a Staphylococcus aureus isolate in cheese[END_REF]Hunt et al., 2015;[START_REF] Valihrach | Production of staphylococcal enterotoxin C in milk[END_REF] and also in the present study the results show to be constant and reliable (see Figure 28 for standard deviations). RT-qPCR was used due to the lack of other methods for enterotoxin measurement, what raises the question if this can be used for approximation of the enterotoxin behaviour. In our case this comparison can just be done for SEG and SEI. The results show that a general tendency to similarities can be seen as reduction due to sodium chloride and higher temperature can be observed for mRNA and protein measurement. On the other side, taking as an example Mu50, this strain seem to have a low expression at mRNA level compared to the other strain, but by looking at the enterotoxin production this is as high as the other ones. Strain 26.1 is exactly the contrary, high expression at mRNA level but under the limit of detection production of SEG and SEI. A reason for these differences could be differences in post-transcriptional processes.

A reason for that could be the different codon usage of the single strains, this means that specific codons and tRNAs concentrations have an effect on the efficiency and fidelity of the translation machinery and in consequence an impact on the amount of protein produced [START_REF] Frumkin | Codon usage of highly expressed genes affects proteome-wide translation efficiency[END_REF][START_REF] Hanson | Codon optimality, bias and usage in translation and mRNA decay[END_REF].

By looking at the mRNA data itself during the cell growth, a general switch on at mid exponential and switch off at late exponential phase can be observed. Differences between the strains are not as markable as the one from the enterotoxin production, were the differences can be predicted, based on the genome as described in our previous study [START_REF] Schwendimann | Staphylococcal enterotoxin gene cluster: prediction of enterotoxin (SEG and SEI) production and of the source of food poisoning based on vSabeta typing[END_REF]. This general of switch on and off was already described by [START_REF] Derzelle | Differential temporal expression of the staphylococcal enterotoxins genes during cell growth[END_REF] and again confirmed in this study. What is new is that translation from mRNA to protein starts immediately, leading to a measurable amount of enterotoxin already after two hours after inoculation.

An open question in the study of egc enterotoxins is if these enterotoxins are all expressed and produced together or if single regulation mechanisms of each enterotoxin will have an influence. The results seem to show that some global regulators are influencing the switch-on and switch-off mechanisms, but that some local promoters are doing a fine tuning.

In literature [START_REF] Kusch | The influence of SaeRS and sigma(B) on the expression of superantigens in different Staphylococcus aureus isolates[END_REF]) σ B was found to be a regulator of egc enterotoxins, as it can be found in front of the egc. The presence if σ B would also mean that enterotoxins can be activated by different stress factors [START_REF] Senn | Molecular analysis and organization of the sigmaB operon in Staphylococcus aureus[END_REF]. In literature it has been described that phage borne se-genes (no studies for egc) are activated by phage inducing agents like low pH, oxidative stress, mitomycin C, NaCl and acetic acid [START_REF] Cao | Elevated enterotoxin A expression and formation in Staphylococcus aureus and its association with prophage induction[END_REF]Sumby and Waldor, 2003a;[START_REF] Wallin-Carlquist | Acetic acid increases the phage-encoded enterotoxin A expression in Staphylococcus aureus[END_REF][START_REF] Zeaki | Assessment of high and low enterotoxin A producing Staphylococcus aureus strains on pork sausage[END_REF].

As a consequence of this information it is questionable if σ B was really activated by applied sodium chloride and temperature. The results of the influence of the stress parameter on the expression and production of enterotoxin showed some differences between the three strains analyzed. The results show that for strain 15SB an increase of enterotoxin expression (for all analyzed enterotoxins) at mRNA level was observed between 5 g/L and 12 g/L, on the other side for all other conditions and strains this could not be observed. The influence of a higher temperature (45 °C) to Mu50 is interesting. For this strain, the growing curve seemed to be more steady at a temperature of 45 °C, on the other side SEI production is increased (15.13 +/-3.3 ng/mL), compared to the other two strains (KS24, 4.03 +/-0.86 ng/mL). The reason for this behaviour can be found in literature, as this strain is a MRSA strain. Literature described a correlation between antibiotic and heat resistance [START_REF] Baldan | Epidemic MRSA clone ST22-IV is more resistant to multiple host-and environment-related stresses compared with ST228-I[END_REF].

Interestingly, for all strains even at 45 °C or 10% sodium chloride, enterotoxins could be detected. In literature no results were found in order to comparable to these findings, as only (Zhao et al., 2017a) performed experiments testing the effect of sodium nitrite, polylysine, chitosan, and tea catechin on cell growth and enterotoxin I production, suggesting tea catechin for inhibition of S. aureus growth and SEI secretion. By comparing these results to the general literature on staphylococcal enterotoxin production it can be observed that egc enterotoxins are produced even at the boundaries of enterotoxin production for sodium chloride (0-10 %) as well as for temperature 10-45 °C (Hennekinne et al., 2012;[START_REF] Tatini | Influence of food environments on growth of Staphylococcus aureus and production of various enterotoxins[END_REF].

Transferring the findings to some more practical application it can be said that the maximal usage of sodium chloride (100 g/L) is not relevant for food production but scientifically interesting. Normal NaCl concentrations in food go until 2300 mg/100 g (meat product such as bacon), in between there is cheese (200-1500 mg/100 g) and fish (600 mg/100 g) [START_REF] Doyle | Sodium Reduction and Its Effect on Food Safety, Food Quality, and Human Health[END_REF], in this range from different food products the two tested NaCl parameter were set (12.5 g/l and 20 g/L).

Conclusion

With the present study we confirm that egc enterotoxins seem to be mostly expressed in the mid-log phase of bacterial growth and seem to switch off at the end of the log phase. What is new is that SEG and SEI are already produced at an early stage of the growing phase, a fact that may be of importance, since this means that is necessary to measure early in a process to control enterotoxin production. In addition also common NaCl and temperature stress, mimicking conditions found in food production, seem not to affect the expression and production of egc enterotoxins up to 20 g/L. However, a diversity between the strains was observed. The study gives insights into the production of egc enterotoxins under stress conditions. This information will enhance the availability of methods controlling egc enterotoxins (SEG and SEI) in food production and storage. Moreover in order to be able to assess the risk in food matrices, further studies should be performed by testing the behaviour of the egc enterotoxins in different kinds of foods like milk, cheese or meat products.

Declaration of interest

None

Acknowledgment

This work was financially supported by the Agroscope research program. The work is a collaboration project between Anses and Agroscope. The development of the ELISA method for G and I was funded in part by the French joint ministerial program of R&D against CBRNE threats. In addition, a particular thanks goes to Simon Briner for the critical reading of this manuscript.

General discussion

The advances in whole genome sequencing and the decrease of the costs for sequencing in the past years lead to an increasing number of sequenced strains. To date sequencing data from 80'000 S. aureus strains can be found on NCBI. In recent years, also the number of strains sequenced coming from foodborne outbreaks are increasing [START_REF] Chieffi | Novel insights into the enterotoxigenic potential and genomic background of Staphylococcus aureus isolated from raw milk[END_REF][START_REF] Merda | NAuRA: Genomic Tool to Identify Staphylococcal Enterotoxins in Staphylococcus aureus Strains Responsible for FoodBorne Outbreaks[END_REF]. This data availability is an opportunity to generate new tools and methods for improving food safety. Large studies with great numbers of sequenced strains can be performed, making it easy to proof specific findings, especially if beside the genome also the isolate is available. Under these circumstances also the prediction of gene expression and protein synthesis, based on genomic data is a field that can be explored. This is the case for S. aureus and its enterotoxins, as to date a lot of outbreaks are of weak evidence. Reason for that are multiple. On one side only for five out of 26 enterotoxins there are commercial detection methods available. In addition, few is known about the so called nonclassical enterotoxins. For this reason new tools and methods such as WGS and new methods for enterotoxin measurements are needed in order to be able to analyse and measure the S.

aureus strains and their enterotoxins and consequently, being able to predict the enterotoxin production based on genome data. With the present study a contribution can be given in this field since a group of the non-classical enterotoxins, the egc enterotoxins were studied at genome level (seg, sei, sem, sen, seo, and seu), at mRNA expression level (seg, sei, sem, sen, seo), and at protein level (SEG and SEI). By comparing the results from the different levels, models to predict SEG and SEI production based on genome data were found. They shed light on new insights into the conditions and limits of enterotoxin production under stress parameters such as NaCl and temperature.

The role of egc enterotoxins

Egc enterotoxins are present in about 50% of S. aureus strains. This percentage has been described in literature [START_REF] Argudin | Genotypes, exotoxin gene content, and antimicrobial resistance of Staphylococcus aureus strains recovered from foods and food handlers[END_REF][START_REF] Jarraud | egc, a highly prevalent operon of enterotoxin gene, forms a putative nursery of superantigens in Staphylococcus aureus[END_REF][START_REF] Smyth | Superantigen genes encoded by the egc cluster and SaPIbov are predominant among Staphylococcus aureus isolates from cows, goats, sheep, rabbits and poultry[END_REF] and was also confirmed again in this study. Very recent studies also describe egc to be the most frequent virulence factor detected in MRSA and MSSA [START_REF] Kwapisz | Presence of egc-positive major clones ST 45, 30 and 22 among methicillinresistant and methicillin-susceptible oral Staphylococcus aureus strains[END_REF]. Beside the high prevalence, these enterotoxins are also quite particular as they are all located on a stretch forming a gene cluster. We observed that this cluster of enterotoxin genes shows no signs of recombination (unpublished data) and they seem to be on a stretch of paralogous genes. In the beginning of this study it was not clear if the gene expression of these enterotoxins is regulated together (single promoter at the start of the egc), or if each gene is regulated separately. With the results achieved in this study we can say that there are some signs indicating a regulation of the whole cluster, but it may be that also the promoters located just before the single enterotoxins also play a role. The whole egc is part of the mobile genetic element vSaβ, which was recently described to be a phage [START_REF] Klaui | Characterization and Comparative Analysis of the Staphylococcus aureus Genomic Island vSabeta: an In Silico Approach[END_REF]. VSaβ was initially a prophage as it is still observed in vSaβ type IV and can still be mobilized. At the time around clone formation, the original vSaβ underwent multiple genetic changes resulting in the different vSaβ types observed today [START_REF] Klaui | Characterization and Comparative Analysis of the Staphylococcus aureus Genomic Island vSabeta: an In Silico Approach[END_REF]).

In addition to the above-mentioned points egc enterotoxins express super antigenic and emetic activity. Emetic activity has been demonstrated on monkeys for SEG, SEI, SEM, SEN and SEO [START_REF] Omoe | Emetic potentials of newly identified staphylococcal enterotoxin-like toxins[END_REF]. As emetic activity has not yet been demonstrated for SEU, this enterotoxin was not taken into consideration in our study. From a food safety point of view, literature [START_REF] Johler | Further evidence for staphylococcal food poisoning outbreaks caused by egc-encoded enterotoxins[END_REF] seems to show the involvement of these enterotoxins in foodborne outbreaks. Recent publications are also describing the super antigenic activity of these enterotoxins and they seem to have an effect particularly on human skin [START_REF] Nowrouzian | Superantigens and adhesins of infant gut commensal Staphylococcus aureus strains and association with subsequent development of atopic eczema[END_REF].

In the past studies of Agroscope (Appendix I) the focus was on genotype B strains (SEA, SED, SEJ and SER) but with the aforementioned facts about the egc enterotoxins the focus of this study was changed to the egc enterotoxins, since not much is known about the expression of these enterotoxins and their involvement in SFPOs in Switzerland and France (Paper I).

vSaβ characterisation and subtyping of egc containing S. aureus

As soon as the WGS from the strain collection used in this study was ready, the characterisation of the vSaβ type was performed, as it seemed to be a good tool to subtype the strains. In addition, [START_REF] Klaui | Characterization and Comparative Analysis of the Staphylococcus aureus Genomic Island vSabeta: an In Silico Approach[END_REF] showed its discriminatory power and the correlation between the vSaβ type and the clonal complex. In this study (Paper I) beside the vSaβ type already described in literature, we describe seven new types. As a consequence we think that, as the diversity of the clonal complex is varying, also the amount of vSaβ type will not be limited to 15. It is supposed that the number of vSaβ types will increase with additionally characterised S. aureus genomes and their vSaβ. By looking at the egc of the different vSaβ types we can see that they differ in the presence/absence of certain enterotoxins (seu), but sometimes also single genes are truncated or fragmented (sen, seg, sem). Also the single egc enterotoxin genes (especially sem, seo and seu) seem to have a large variability (sometimes < 90%

similarity at amino acid level, Paper I), which was also described by Mérda et al. (2020). Strains having the same type of vSaβ have a similarity of 100% at amino acid level between them for all egc enterotoxins and the whole egc. In the past other subtyping methods for the egc cluster were used and described in literature (egc type 1-6) [START_REF] Chieffi | Novel insights into the enterotoxigenic potential and genomic background of Staphylococcus aureus isolated from raw milk[END_REF]. With these methods different types of enterotoxins and their variants were used to define the egc type. Based on the new findings of this study we consider this method as too complicated (because of the different variants described) and with insufficient discriminatory power. Consequently, we propose to use the vSaβ typing as a tool to distinguish the different egc containing strains, when it considers this part of the genome as a whole and not the single enterotoxins. Using the here described method would also help to reorganise the pretty confusing egc nomenclature in use. Instead of using different variants like SElU1, SElU2, SElW simply the type of vSaβ could be used. In addition, in some literature the presence of SElV in the egc was described. This enterotoxin was not found in any of the genomes analysed in this study (Paper I) nor in the 156 genomes analysed by Mérda et al. (2020), which raises the question of the relevance of this enterotoxin. Overall I think that staphylococcal enterotoxins nomenclature and classification should be revised and newly organized since every year more studies are published describing new enterotoxins. In any case it should clearly be defined which of them are only variants of already described enterotoxins and which are completely new ones. This should be based on phylogenetic studies using the new bioinformatic tools developed in the recent years.

Prediction of enterotoxin production

After having characterized and allocated the strains to the 15 different types of vSaβ, the question raised if some clear differences could also be observed in enterotoxin production. At the time of the study an ELISA method for detection of SEG and SEI was available and by applying this method a subset of the strains (approx. three strains per vSaβ type) was analysed for their production of SEG and SEI (Paper I). We were surprised by the clear results as it could be observed and statistically demonstrated that some vSaβ types produced a "high" amount of SEG and SEI enterotoxins and others produced a "low" amount of them. These results and the correlation between clonal complex and vSaβ enabled us to create a tool to predict SEG and SEI enterotoxins based on WGS data. However, the reason for the existence of this correlation is not yet clear. We assumed that differences at the promoter level are responsible for it, depending on the vSaβ type. Deeper analysis of the promoter of the enterotoxins (data not included in this manuscript) showed different knowledge gaps due to the unknown TSS (transcriptional starting site) of the enterotoxins. Without knowing this site, we concluded that only presumptive promoters can be allocated and a comparison of presumptive promoters makes a comparison between vSaβ types difficult. A definition of TSS can only be done by performing RNA sequencing, what I assume needs to be done in future.

Interestingly is also the fact that the two analysed enterotoxins (SEG and SEI) seem to follow the same expression pattern: when SEG is produced "high" also SEI is produced "high" and the same pattern for the low production level. The produced quantities of SEI are much higher than those produced of SEG. Due to this fact and due to the strong link between vSaβ and the clonal complex, I assume that there are some global regulation systems for the different egc enterotoxins in the vSaβ or somewhere else on the genome. They are probably correlated to the clonal evolution. Also with the approach of measuring the protein only, it was not clear if the differences in enterotoxin production are caused by differences in transcription or translation. In order to obtain more clarity in this question, the expression of the egc enterotoxins was measured.

Correlation between egc enterotoxin expression and production

Expression of egc enterotoxins was measured for a subset of strains using RT-qPCR (Paper II). A method for the measurement of enterotoxin expression was developed for seg, sei, sem, sen and seo. As described in past studies (e.g. Derzelle et al. 2014), we confirmed that expression of egc enterotoxins seems to be highest at the mid exponential growing phase.

This fact confirms that egc enterotoxins are non-agr dependent enterotoxins as otherwise they would have been expressed at a later point in the late exponential growing phase. Also at protein level SEG and SEI were surprisingly already measurable and quantifiable two hours after inoculation. For the analysis of the expression a subset of strains was chosen based on three criteria: 1) diverse in the genome, 2) different patterns of enterotoxin production (Paper I), and 3) were involved in foodborne outbreaks. Contrary to our expectations for the selected strains, no differences could be observed at the expression (mRNA) level between the strains, as the ones producing very low amounts of enterotoxin -G and I -had the same expression (mRNA) as the one producing high amounts of enterotoxins G and I. With these results I'm convinced that the egc enterotoxins are all expressed independently of the vSaβ type, but when it comes to translation from mRNA to protein a diversity in vSaβ/CC seems to play a role.

In literature, reasons for differences in post-transcriptional steps are described to rely on translation efficiency. There are multiple causes for differences in translation efficiency such as differences in mRNA structure, differences in the Shine-Dalgarno sequence or regulation by some other RNA transcripts [START_REF] Chiaruttini | On the role of mRNA secondary structure in bacterial translation[END_REF][START_REF] Li | How do bacteria tune translation efficiency?[END_REF][START_REF] Meyer | The role of mRNA structure in bacterial translational regulation[END_REF]. As there are some variations in the single enterotoxin genes between the different vSaβ I think that this could lead to some differences in RNA structure or in the Shine-Dalgarno sequence and therefore, leading to some differences in translation efficiency. Also the difference between SEG and SEI could be explained as a difference in translation efficiency. From a transcriptional point of view, as there are just minor differences between the single enterotoxins it is not yet clear if enterotoxin genes are all regulated by the same promoter, some single promoters or a combination of both.

No general conclusions on enterotoxin production can be made for enterotoxins for which a protein measurement method is not yet available like SEM, SEN and SEO: also in this case it could be that they are expressed, but maybe not translated into proteins. From literature we only know that SEM protein could be measured in strains from foodborne outbreaks (Zhao et al., 2017b).

Importance of egc enterotoxins for food safety

When starting this study it was not clear if egc enterotoxins play a relevant role in foodborne outbreaks. Only a few studies showed the possible involvement of these enterotoxins in SFPOs [START_REF] Johler | Further evidence for staphylococcal food poisoning outbreaks caused by egc-encoded enterotoxins[END_REF][START_REF] Umeda | Molecular and epidemiological characterization of staphylococcal foodborne outbreak of Staphylococcus aureus harboring seg, sei, sem, sen, seo, and selu genes without production of classical enterotoxins[END_REF]. In this study (Paper I), the strains involved in SFPO showed their ability to produce SEG and SEI and even in "high" amounts.

But it has to be noted that "high" amounts in this study, especially for SEI, are still much lower than the ones measured for SEA [START_REF] Zeaki | Evaluation of Potential Effects of NaCl and Sorbic Acid on Staphylococcal Enterotoxin A Formation[END_REF]. Also the emetic activity is much higher (lower amounts needed) for SEA rather than for SEI [START_REF] Ono | The emetic activity of staphylococcal enterotoxins, SEK, SEL, SEM, SEN and SEO in a small emetic animal model, the house musk shrew[END_REF]. These facts raise the question if a single egc enterotoxin can be the cause for a foodborne outbreak or if it is the cumulative effect of different enterotoxins produced that causes foodborne outbreaks, because they potentially increase the severity of its single effects on human health. This is supported by the fact that all egc enterotoxins seem to be expressed and potentially also produced together (Paper II). In addition and as mentioned in the previous chapter, the egc enterotoxins seem to be produced very fast (2 hours after inoculation), also under higher temperatures (45 °C) and high NaCl concentration (up to 100 g/L). These enterotoxins can be produced in a very wide range of conditions which are normally considered as critical for growing and enterotoxin production. Obviously there is a big impact for the production of cheese. Finally and not to underestimate is the fact that egc seems to be present in approx. 50% of the strains. These findings support the fact that egc enterotoxins may play a role in foodborne outbreaks. At the moment an egc enterotoxin caused SFPO would fall into the category of weak evidence outbreak, as no laboratory would be able to measure egc enterotoxins from the strains nor in the food. The inability of measuring these and other staphylococcal enterotoxins is controversy to the fact that according to the Swiss and European law Staphylococcal enterotoxins should not be present in foods. A very disappointing situation that should be solved by further research and invention.

Ideas for outbreak investigation with suspected involvement of egc enterotoxins

Considering the fact that egc enterotoxins seem to be more involved in SFPO than expected also new strategies and tools should be developed to determine egc enterotoxins caused outbreaks. As already mentioned in the previous chapter, in my opinion the whole egc should be considered as a unity from an analytical and epidemiological point of view. This perspective would simplify the analytical procedure and the full investigation of egc enterotoxin caused outbreaks (Figure 31). Linking together all new insights of this study leads to a suggestion on how an egc caused outbreaks could better be clarified in the future. Consequently, there is a high probability that information on the CC found is already known.

With this comparison to known CCs additional information will be available (Paper I): 1) It is possible to know if the strain is a "high" or "low" enterotoxin producer. If it appears that e.g. the strain is a CC705 (having vSaβ IV and being a low producer) it is clear that in this case egc enterotoxins would not have been involved in the outbreak. In the case of a CC45 the contrary could be assumed. 2) A CC can give an indication of the possible source of the involved S.

aureus strain. Again, assuming it is CC705, even when SEG and SEI enterotoxins are not involved, with the new insights we can assume that the source of this strain is cattle. On the other hand, a strain being CC45 is most probably from human origin. By knowing this information, the possible source of contamination can more easily be found and eliminated.

If the results of the sequencing show a CC where neither enterotoxin production studies nor vSaβ characterization studies have been performed, no information on prediction or source can be given. In this case these SEG and SEI production studies should be performed with the isolated S. aureus strain and vSaβ should be characterized. Applying these methods the data base will continuously be enlarged and the cases of unknown CC will become rare. In addition, if in the future methods for the detection of SEM, SEN and SEO are available, these enterotoxins show maybe the same pattern and CC dependency as SEG and SEI. For a strong evidence outbreak proof, the enterotoxins need to be detected in the food matrix and also evidence that the isolated strain is able to produce these enterotoxins. With the new information of this study, in future it might be necessary to only analyze one enterotoxin (e.g. SEI) instead of all of them (SEG, SEI, SEM, SEN, SEO).

If the S. aureus strain is not directly going to be sequenced, qPCR for the detection of enterotoxins needs to be performed. In such a situation, I suggest to only use one qPCR for the detection of sei. Here again the new insights of egc should be used: As we know that sei is always present on each vSaβ type, not being truncated and having a low variability (Paper I, [START_REF] Klaui | Characterization and Comparative Analysis of the Staphylococcus aureus Genomic Island vSabeta: an In Silico Approach[END_REF], this enterotoxin gives an indication of the presence or absence of egc. If egc is present, CC should be identified by PCR and sequencing of the seven housekeeping genes. Afterwards the procedure is the same as described in the case of sequencing of the strain.

Conclusions and perspectives

The main goal of this study was to find a model that can predict staphylococcal egc enterotoxin production based on WGS data. This goal has been reached and as a side effect some more insights on egc enterotoxins and vSaβ have been found.

The correlation between clonal complex and vSaβ type, together with the fact that enterotoxin G and I production is dependent on the vSaβ type and can statistically be allocated to "high"

and "low" production, made it possible to predict SEG and SEI production based on whole genome data. In addition, the new insights also show that based on the CC indication the possible source of contamination can be determined. This new knowledge can be used in SFPOs cases in order to evaluate the involvement of a specific strain in an outbreak.

Beside the new insights, I nevertheless think the prediction model needs to be further extended due to the diversity of clonal complexes and by genotypical and phenotypical characterisation of the not already described CCs. This should be done by analysis of the vSaβ and the test for the production of enterotoxins G and I. In addition, once a quantitative method for detection of enterotoxin SEM, SEN and SEO is available the strains of the different vSaβ types should be analysed for the production of these enterotoxins. If the results also give the same insights as the one for SEG and SEI, the model could be extended to all egc enterotoxins.

In the beginning the reason for the differences in the correlation of vSaβ and enterotoxin production were assumed to be in the diversity of the promoters. Due to the absence of information about the TSS, promoter studies could not be performed as planned. In consequence, I think that RNAseq would be needed to be able to define the TSS. In addition, with this method it could also be confirmed if egc enterotoxins are all expressed together or singularly.

From the point of view of food safety also some new insights into production of SEG and SEI under different parameters were gained with this study. Under the stress parameters used here, expression (mRNA) was measured for all egc enterotoxins. Surprisingly, SEG and SEI production of these enterotoxins could already be measured two hours after inoculation, with an incubation temperature of 45°C or NaCl concentrations up to 100 g/l. These results show that SEG and SEI can also be produced under harsh conditions. Therefore, it should be further observed how they are produced in a food matrix. Especially with foods where a lot of human handling is required (e.g. artisanal cheese making and similar), challenge tests should be performed in order to find out if these enterotoxins have the ability to be produced under real life conditions. Nevertheless, statements at the protein level cannot be made for SEM, SEN and SEO, as we have no knowledge if translation from mRNA to protein happened for these enterotoxins.

The enterotoxin gene cluster is composed of five enterotoxin genes for which emetic activity is demonstrated. In my opinion this cluster should be considered as one and in the future, when more knowledge will be available about the single enterotoxins and their behaviour, maybe only one representative enterotoxin out of the five can be used for demonstrating the implication of an egc positive S. aureus strain in a SFPO. Before reaching this level, a detection method should be made available for all five enterotoxins in order to be able to study the complete toxin production pattern and to see if the total amount of enterotoxin produced correlates with the high implication of egc enterotoxins in SFPOs.

Conclusions et perspectives (French)

L'objectif principal de cette étude était de trouver un modèle qui puisse prédire la production

List of tables

List of figures

Introduction

Coagulase-positive staphylococci (CPS) are foodborne pathogens with an ability to produce staphylococcal enterotoxins (SEs) [START_REF] Kummel | Staphylococcus aureus Entrance into the Dairy Chain: Tracking S. aureus from Dairy Cow to Cheese[END_REF]. According to European Food Safety Authority [START_REF]The European Union summary report on trends and sources of zoonoses, zoonotic agents and food-borne outbreaks in 2017[END_REF],

SEs are a major cause of foodborne illness in humans, and many studies have reported the role of SEs in foodborne outbreaks (20,[START_REF] Schmid | Outbreak of staphylococcal food intoxication after consumption of pasteurized milk products, June 2007[END_REF][START_REF] Suzuki | Food poisoning outbreak in Tokyo, Japan caused by Staphylococcus argenteus[END_REF]. Although staphylococci are killed during food processing, SEs remain viable because of their high thermal stability [START_REF] Loir | Staphylococcus and food poisoning[END_REF]. In contrast to tests aimed at determining the viability of CPS, tests for ascertaining the viability of SEs are demanding and relatively expensive. Therefore, they are usually not performed. Moreover, currently available enzyme-linked immunosorbent assay kits for SEs are not able to detect all known SEs (4). In addition, detection of different enterotoxin genes in staphylococcal strains using the polymerase chain reaction assay does not always provide accurate information on enterotoxin gene expression [START_REF] Hennekinne | Staphylococcus aureus and its food poisoning toxins: characterization and outbreak investigation[END_REF][START_REF] Hummerjohann | Enterotoxin-producing Staphylococcus aureus genotype B as a major contaminant in Swiss raw milk cheese[END_REF][START_REF] Wu | A Review of the Methods for Detection of Staphylococcus aureus Enterotoxins[END_REF].

The foods involved in foodborne outbreaks mainly include ready-to-eat products, often meat and dairy products [START_REF] Hennekinne | Staphylococcus aureus and its food poisoning toxins: characterization and outbreak investigation[END_REF]. Among dairy products, raw milk cheese is most susceptible to contamination with SEs ( 6). The enterotoxins in cheese often originate from raw milk from cows with Staphylococcus aureus mastitis [START_REF] Schmid | Outbreak of staphylococcal food intoxication after consumption of pasteurized milk products, June 2007[END_REF]. However, poor food safety practices and poor personal hygiene can be a source of contamination [START_REF] Hennekinne | Staphylococcus aureus and its food poisoning toxins: characterization and outbreak investigation[END_REF].

Using the ribosomal spacer PCR (RS-PCR) method, more than 100 genotypes and variants of S. aureus isolated from bovine inframammary infections have been identified, 17 subtypes of which were genotype B (GTB) and genotype C (GTC), which represented 81% of the isolates [START_REF] Fournier | Bovine Staphylococcus aureus: association of virulence genes, genotypes and clinical outcome[END_REF]. The remaining genotypes were rare and accounted for only 1.0-4.0% of all isolates [START_REF] Fournier | Bovine Staphylococcus aureus: association of virulence genes, genotypes and clinical outcome[END_REF].

Staphylococcal genotypes are highly associated with virulence gene patterns [START_REF] Cosandey | Staphylococcus aureus genotype B and other genotypes isolated from cow milk in European countries[END_REF][START_REF] Fournier | Bovine Staphylococcus aureus: association of virulence genes, genotypes and clinical outcome[END_REF] . Various enterotoxin genes, including sea, sed, sej, and ser, have been identified in S. aureus GTB (4).

This genotype was also shown to persist along the food chain and its enterotoxin genes were expressed and linked to foodborne poisoning outbreaks involving raw milk cheese [START_REF] Hummerjohann | Enterotoxin-producing Staphylococcus aureus genotype B as a major contaminant in Swiss raw milk cheese[END_REF][START_REF] Kummel | Staphylococcus aureus Entrance into the Dairy Chain: Tracking S. aureus from Dairy Cow to Cheese[END_REF]. In contrast, S. aureus GTC was typically positive for sec, seg, sei, and tst, [START_REF] Fournier | Bovine Staphylococcus aureus: association of virulence genes, genotypes and clinical outcome[END_REF][START_REF] Graber | Mastitis-related subtypes of bovine XVIII Staphylococcus aureus are characterized by different clinical properties[END_REF] and was not V linked to outbreaks traced to raw milk cheese [START_REF] Hummerjohann | Enterotoxin-producing Staphylococcus aureus genotype B as a major contaminant in Swiss raw milk cheese[END_REF]. For the remaining genotypes, the virulence gene pattern was heterogeneous [START_REF] Fournier | Bovine Staphylococcus aureus: association of virulence genes, genotypes and clinical outcome[END_REF], and a few genotypes were associated with staphylococcal food poisoning [START_REF] Hummerjohann | Enterotoxin-producing Staphylococcus aureus genotype B as a major contaminant in Swiss raw milk cheese[END_REF].

The clinical properties of S. aureus exhibit clear differences, depending on the genotype [START_REF] Graber | Mastitis-related subtypes of bovine XVIII Staphylococcus aureus are characterized by different clinical properties[END_REF].

In the case of Staph. aureus GTB, up to 87% of cows per herd showed inframammary infection, therefore, S. aureus GTB is a contagious mastitis pathogen, which spreads easily among cows [START_REF] Fournier | Bovine Staphylococcus aureus: association of virulence genes, genotypes and clinical outcome[END_REF][START_REF] Graber | Mastitis-related subtypes of bovine XVIII Staphylococcus aureus are characterized by different clinical properties[END_REF][START_REF] Van Den Borne | A longitudinal study on transmission of XX Staphylococcus aureus genotype B in Swiss communal dairy herds[END_REF]. In contrast, S. aureus GTC and most of the other genotypes were detected only in individual cows with mastitis [START_REF] Cremonesi | Genomic characteristics of Staphylococcus aureus strains associated with high within-herd prevalence of intramammary infections in dairy cows[END_REF][START_REF] Fournier | Bovine Staphylococcus aureus: association of virulence genes, genotypes and clinical outcome[END_REF][START_REF] Graber | Mastitis-related subtypes of bovine XVIII Staphylococcus aureus are characterized by different clinical properties[END_REF]. In previous research, S. aureus GTC was isolated from milk of cows with inframammary infections throughout Europe, whereas S. aureus GTB was detected in milk from cows with inframammary infections only in countries bordering Switzerland [START_REF] Cosandey | Staphylococcus aureus genotype B and other genotypes isolated from cow milk in European countries[END_REF]. Other genotypes, except GTR, were rarely detected, although some of the genotypes were determined increasingly in specific countries (4).

In cheese production, growth of S. aureus can occur prior to processing, as the raw milk used in production may be stored at 8-18° C for up to 24 h before use. During cheese production, temperature, pH, competitive pressure from starter flora, and lactose starvation influence the growth of S. aureus and enterotoxin formation. Salt stress may also play a role in S. aureus growth and enterotoxin formation, although in hard and semi-hard Swiss cheeses, CPS counts start to decline during pressing or a few days after brining when the salt concentration in the cheese is still low [START_REF] Bachmann | The fate of potentially pathogenic bacteria in Swiss hard and semihard cheeses made from raw milk[END_REF]. Interactions between these parameters may enhance enterotoxin production [START_REF] Duquenne | Milk maturation temperature and time are key technological parameters to limit staphylococcal enterotoxin production during uncooked semi-hard cheese manufacture[END_REF][START_REF] Jakob | Leitlinie für die gute Verfahrenspraxis bei der Milchgewinnung und -verarbeitung in Sömmerungsbetrieben [Guideline for good practice in milk production and processing in summer mountain farms[END_REF][START_REF] Walcher | Staphylococcus aureus reservoirs during traditional Austrian raw milk cheese production[END_REF].

According to Commission Regulation (EC) No. 2073/2005 on microbiological criteria for foodstuffs (1), staphylococcal tests of cheese must be conducted at the time when it is assumed that the concentration will be highest. For hard and extra-hard cheese, this is usually before the process step "scalding" occurs. If the CPS/g concentration exceeds 10

Materials and Methods

Strains and inoculum preparation

For the inoculation of raw milk, five different S. aureus strains (a-e) were used, all of which had been previously isolated from cheese and analysed by Hummerjohann et al. 2014 (15) (Table 1). Some of the strains, such as strain b, were previously reported to be involved in a staphylococcal food poisoning outbreaks (SFPOs) [START_REF] Graber | Mastitis-related subtypes of bovine XVIII Staphylococcus aureus are characterized by different clinical properties[END_REF]. All strains used were stored at -20 °C and revived on a Colombia agar and 5% sheep blood (BioMérieux, Geneva, Switzerland). For the preparation of the inocula, one colony of each strain (a-e) grown on Columbia agar and 5% sheep blood (was transferred into 5 ml of Oxoid Staphylococcus medium No. 110 (Thermo Fisher Scientific, Pratteln, Switzerland) and incubated for 18 h at 37° C. These cultures were then diluted (0.9 % NaCl) to obtain inocula containing about 10 5 CFU/ml and stored at -20° C until use (maximum time of storage 3 months). Before use, the bacterial count of the inocula was determined by plating on Columbia agar and 5% sheep blood. This method was used in order to have the most exact concentration of the inocula. SEs were detected using a mini Vidas SET2 (BioMérieux) according to the manufacturer's instructions.

Cheese production and experimental set-up

A laboratory cheese model system (Supplementary Material Figure . S1) was set-up that complied with the parameters and conditions (temperature profile, pH, culture, rennet) encountered during the first 24 h of the cheese-making process. In the model system, cheese was produced in a 250-ml Schott bottle containing an initial volume of 200 ml of raw milk. The raw milk was sourced from a local cheese dairy and was also tested for the presence/absence of CPS.

In each batch, five cheeses inoculated with S. aureus (strains a-e) and one negative control were produced at the same time, with one scalding temperature step. The model system, in which six Schott bottles (with stir bars) were placed in a water bath in which the temperature was controlled by a thermostat (Lauda RE310; Lauda Ecoline Staredition E300; LAUDA-Brinkmann, Delran, NJ, USA). The water bath was stirred using a Multi-position magnetic stirrer (Variomag, Daytona Beach, FL, USA) using an agitator speed of 220 rpm. Five of the bottles (containing strains a-e) were kept closed. The sixth bottle was used to record the temperature during curdling and scalding. The water bath was covered with floating balls to prevent evaporation. 2. were added, and the milk was left for 30 min for prematuration. Subsequently, 50 μl of calf rennet (Lab Winkler GR orange; Winkler AG, Konolfingen, Switzerland) were added to induce mild coagulation, and the coagulum was cut 40 min later using a kitchen knife, aiming for a curd size of 5 mm. This was reached by cutting the curd horizontally, then rotating the bottle IX in the hand aiming at also having a vertically cut of the curd. The resulted curd (size and homogeneity) cannot be compared to the commercial practices but this should not have had any impact on the outcome of the experiment. The curd was then heated to scalding temperature while stirring gently. The temperature-time profiles applied were comparable to those used in commercial cheese production (Table 2). After scalding, the whey and curd were separated using kitchen sieves. The remaining curd was then placed in two 50 ml Falcon tubes and centrifuged for 15 min at 3,000 rpm (Heraeus Megafuge 16 R; Thermo Scientific). Thereafter, the remaining whey was removed, the cheese was weighed (approximately 20 g), and the tubes were immediately returned to the water bath, which was still at a scalding temperature. The experimental cheeses were then cooled to 26°

VIII

C for 20 h. The temperatures applied were the same as those applied in artisan cheese factories. Therefore, these temperature-time profiles reflected the situation in the peripheral zones of the cheese samples.

In each batch, six experimental cheeses, of which five contained one of the five S. aureus strains, were produced under different scalding temperatures (48, 50, 52, 54, or 56° C). Each batch was repeated five times, resulting in 25 batches, 125 inoculated cheeses, and 25 control cheeses. The temperature and pH were measured in the control cheeses using a specific measuring equipment (Ahlborn Almeno 2590; Ahlborn, Holzkirchen, Germany).

X

Microbial analysis and SE detection

The raw milk [M], raw milk with inoculum [A], curd [S], and cheese [K20] samples for microbiological examinations were obtained as illustrated in Figure 1. S. aureus cell viability in the samples was analysed using the Tempo STA method (BioMérieux). This method was previously validated in house against the ISO 6888-3:2003 (17) method. SEs were detected only in the K20 samples using the ISO 19020:2017 (18) method and a Vidas SET2 immunoenzymatic assay (BioMérieux). Instead of 25 g sample material as required by ISO 19020:2017, only 5 g of cheese were analyzed due to the miniaturized system. All the reagents used were proportionally adapted, without affecting the limit of detection.

Statistical evaluation

For statistical analysis, the student's T-test and an analysis of variance were applied using

Microsoft Office 2016 Excel (Microsoft, Redmond, WA, USA). This model accounted also for repeated measurements. The p-value threshold for statistical significance was 0.05. Data analysis were performed to check for significant difference in bacterial concentrations between the strains (a-e) used (always taking into account the five measurements), between the different scalding temperatures (46-56 °C) and the changing concentrations of CPS between the different production steps (raw milk, curd and cheese).

Results and Discussion

Model system for cheese production

As there is no pilot plant in Switzerland for large-scale experiments with class 2 microorganisms, the present study used a laboratory cheese-making model. During the development of the cheese-making model, care was taken to ensure that the cheese-making parameters (temperature and pH) corresponded to those used in Alpine cheeseries (Supplementary Material Figure S2-S3). This goal was achieved by using a microprocessorcontrolled water bath and continuously recording the process parameters (pH and temperature) during each experiment. As the parameters and results were reproducible on XI different experimental days, the system used can be considered stable and reliable. In addition, the temperature and pH of the laboratory cheeses were comparable to those of hard and semihard Swiss cheeses found in Alpine cheeseries (Tete de Moine and Gruyere were taken as reference based on cheese consultancy experiences and literature [START_REF] Hartmann | Hard cheeses[END_REF]). As an average a pH of 5 +/-0.04 was recorded in all the cheese batches after 20 h which corresponds to the pH of 5 found in alpine cheeseries for the production of Tete de Moine and Gruyere. The pH was measured in the sample without inoculum, but since the concentration of Staphylococcus aureus (depending on the scalding temperature 10 2 -10 7 CFU/ml) is much lower than the one of the starter culture (assumed to be ~10 9 CFU/ml), it will not influence the pH. All raw milk used was tested negative for CPS, and Staphylococcus aureus coming from a natural contamination could therefore be excluded.

Effect of scalding temperatures on S. aureus concentrations and SE production

The time course in increases in S. aureus concentrations during cheese production was independent of the strain used, with no significant difference found in the concentrations of the different strains for the measurements at the time points "raw milk" and "curd"(P > 0.05). Each batch showed an increase of up to two log steps (Figure 2). This increase was attributed to the physical entrapment in the curd, as reported previously in the literature [START_REF] Tatini | Production of Staphylococcal Enterotoxin A in Cheddar and Colby Cheeses[END_REF]. The initial concentration in the inoculated raw milk (approximately 10 4 CFU/ml) was always the same, with no significant differences (P > 0.05) observed what was also the case for the concentration in the curds (approximately 10 6 CFU/ml, P > 0.05), However, after 20 h, there were significant differences in the concentration of S. aureus in the cheese, both between the different strains (P = 0.02) and between the different scalding temperatures (P = 0.0006) (Figure 2). For example the concentration of S. aureus strain d increased notably up to a range of 6 log to 7 log CFU/g (Fig. 2) even after applying a scalding temperature of 50 or 52° C.

Based on the cell counts, the viability of strains a, c, and e clearly declined after 20 h following XIII exposure to scalding temperatures of 50° C or higher and fell to < 100 CFU/g at a scalding temperature of 56° C (Fig. 2). It has been assumed that CPS are inactivated or damaged to such an extent that toxin production in cheese is no longer possible after scalding at 52° C. This was the case for strain d (Table 3), where SEs were produced up to a scalding temperature of 50° C. At 52° C, toxin concentrations were near the limit of detection and not detectable at 54 and 56° C.

Unexpectedly, for strains b and c, SEs were produced up to a scalding temperature of 56° C, whereas strains a and e did not produce enterotoxins even at 46° C (Table 3). Strain-specific differences in SE production by S. aureus have been described previously in the literature [START_REF] Duquenne | Milk maturation temperature and time are key technological parameters to limit staphylococcal enterotoxin production during uncooked semi-hard cheese manufacture[END_REF].

The strains used in the present study have genotypic and phenotypic differences: strains b, c and d are S. aureus GTB [START_REF] Cremonesi | Genomic characteristics of Staphylococcus aureus strains associated with high within-herd prevalence of intramammary infections in dairy cows[END_REF][START_REF] Graber | Mastitis-related subtypes of bovine XVIII Staphylococcus aureus are characterized by different clinical properties[END_REF], whereas strains a and e are GTC and GTO, respectively.

GTB is associated with contagious mastitis and causes herd problems instead of single animal problems like GTC or GTO [START_REF] Cremonesi | Genomic characteristics of Staphylococcus aureus strains associated with high within-herd prevalence of intramammary infections in dairy cows[END_REF][START_REF] Fournier | Bovine Staphylococcus aureus: association of virulence genes, genotypes and clinical outcome[END_REF][START_REF] Graber | Mastitis-related subtypes of bovine XVIII Staphylococcus aureus are characterized by different clinical properties[END_REF][START_REF] Sartori | Sanitation of Staphylococcus aureus genotype B-positive dairy herds: A field study[END_REF][START_REF] Van Den Borne | A longitudinal study on transmission of XX Staphylococcus aureus genotype B in Swiss communal dairy herds[END_REF]. In addition, this genotype is able to persist in the food chain [START_REF] Kummel | Staphylococcus aureus Entrance into the Dairy Chain: Tracking S. aureus from Dairy Cow to Cheese[END_REF]. The persistence of this genotype and its adaption to the dairy environment, including elevated temperatures, would explain the survival and enterotoxin production of this XIV genotype in the present study. Heat adaptation to a similar temperature as the one used in this study (58 °C) was already described in literature [START_REF] Cebriàn | Heat-adaptation induced thermotolerance in Staphylococcus aureus: Influence of the alternative factor σB[END_REF] In terms of SE production, it should be noted that not all five strains produce the same enterotoxins. Strain a produces staphylococcal enterotoxin C (SEC), strain c produces SEA, strains d and e produce SED, and strain b produces SEA and SED. According to the literature, not all strains begin to produce enterotoxins at the same CPS concentration [START_REF] Hunt | Modelling production of S. aureus enterotoxin Cbovine in milk, and its production during cheesemaking[END_REF]. In line with the Hygienic Regulation (1), enterotoxin detection only has to take place when the staphylococci concentration reaches 10 5 CFU/g. Previous research reported only SEA production at this concentration, with SEC produced only at a S. aureus concentration of 10 8 CFU/g [START_REF] Hunt | Modelling production of S. aureus enterotoxin Cbovine in milk, and its production during cheesemaking[END_REF]. The aforementioned explains why SEs are not detected in the presence of strain a. However, in the present study, SE production occurred at S. aureus concentrations lower than 10 5 CFU/g. The presence of enterotoxin in the inoculum can be ruled out, as this was checked at the time of the inoculum preparation. The following factors may explain the finding:

1) It is possible that strains b and c are generally good SE producers and able to produce toxin outside the optimal temperature range between 10 and 48° C [START_REF] Tatini | Production of Staphylococcal Enterotoxin A in Cheddar and Colby Cheeses[END_REF] and possibly even at low concentrations (< 10 5 CFU/g), since in curd for a short time this concentration was reached (due to the physical enrichment) but scalding temperature was immediately applied and decreased the concentration of CPS, in consequence no enterotoxin production was possible in this short time Thus, SEs could have been produced in the heating phase and/or after scalding in the 20 h of cooling, as was found for strain b (56° C scalding temperature) where a S. aureus concentration of 10 3 CFU/g was detected after 20 h. This idea is supported by the findings of Johler et al. (2015) (20), who described enterotoxin formation in ripened cheese at a S. aureus concentration of 10 3 CFU/g. 2). The finding of SE production at lower S. aureus concentrations in the present study may also be explained by strains b and c having the ability to reach the log phase very quickly and thus achieve a concentration of > 10 5 CFU/g during heating to the scalding temperature and produce enterotoxins in this phase. There are no studies in the literature to confirm or refute this idea. In this study a thawed inoculum instead of an overnight culture was used, what may have had an impact on the duration of the lag XV phase of the bacterial growing. However, extrapolating the results from literature [START_REF] Medvedová | Growth Characterisation of Staphylococcus aureus in Milk: a Quantitative Approach[END_REF] it seems that the concentrations reached are comparable to an overnight culture. A longer lag phase, due to the additional stress of the bacteria due to the thawing would mean that in this study the "good case" was used, on the other side it is questionable if S. aureus in the milk would be as fit as in an overnight culture. Duquenne et al. (2016) (9) reported that temperature and time were key parameters in controlling SE production during the cheese-production process.

However, the temperatures (between 32 and 38° C) and times (15 to 45 min) used in their study were markedly different to those applied in the present study. This may explain the enterotoxin production in their study. With the parameters used in the present experiment, there should be no risk of increased SE production. We conclude that milk prematuration for less than 30 min and a temperature below 35° C are sufficient to minimize the risk of enterotoxin production.

Previous research reported that strains, such as the commonly used Lactococcus lactis, from starter cultures added to milk may affect S. aureus growth and viability [START_REF] Cretenet | Staphylococcus aureus virulence and metabolism are dramatically affected by Lactococcus lactis in cheese matrix[END_REF]. However, if the scalding temperature exceeds 45° C, mesophilic starters are inappropriate. Therefore, in our study, we used a thermophilic starter (RMK 150) containing Lactobacillus delbrueckii spp.

lactis and Streptococcus thermophilus.

Based on the results and taking into consideration that a lab model system, using a specific starting concentration of 10 4 CFU/ml, being able to reproduce the temperature curve in the peripheral zone of a real cheese (with specific parameters for Gruyere and Tete de Moine) was used, the initial hypothesis that S. aureus in Swiss Alpine hard and extra-hard raw milk cheese production posed no risk of SFPOs was not confirmed. Unexpectedly, the results demonstrated that specific strains can produce SE even in a process with scalding temperatures above 52° C, particularly S. aureus GTB, which is common on Swiss Alps seem to have this ability.

In this study SE production was studied only under very specific conditions (concentration, curd size, model size and parameters). To get more reliable results there is need to further XVI study the behaviour of S. aureus and its mechanism of enterotoxin production in the cheese food matrix. Especially there is need for studies using real-size cheese that are not only mimicking the peripheral zone of a cheese and by using different cheese production parameters and additional starting concentrations (especially lower ones like 10 2 -10 3 CFU/ml).

In addition, it would also be interesting to further study why there is the difference between the strains and particularly why S. aureus GTB seems to behave so differently. Beside the infection in human beings and animals, Staphylococcus aureus can produce different enterotoxins in food. These enterotoxins can cause vomiting and diarrhea, often involving many people and causing so called staphylococcal foodborne outbreaks (SFPO). To date a lot of SFPO are of weak evidence. One reason for this could be, that currently only five, so called classical enterotoxins, out of 26 known staphylococcal enterotoxins can be analyzed using commercially available kits. A subset of the non-classical enterotoxins (seg, sei, sem, sen, seo and seu) is located on the same enterotoxin gene cluster (egc) which is part of the S. aureus genomic island vSaβ. These enterotoxins have been described as likely being involved in SFPOs.

XXIX

New tools such as whole genome sequencing and new methods for enterotoxin measurement are needed in order to be able to analyse the S. aureus strains and their enterotoxins and, consequently, being able to predict the enterotoxin production based on genome data. This was the main aim of this work.

In order to do so, the egc enterotoxins were studied at genome (seg, sei, sem, sen, seo, and seu), at mRNA (seg, sei, sem, sen, seo), and at protein level (SEG and SEI).

We demonstrate that the in vitro production of SEG and SEI can be predicted based on the vSaβ-type and the clonal complex (CC) of a strain. Furthermore, the vSaβ-type/CC enables the prediction of the source of an egc-positive SFPO strain (animal or human derived).

Enterotoxin production (SEG and SEI) under stress conditions was also tested, showing that common salt concentrations and temperature stress, mimicking conditions found in food production, seem not to affect the production of egc enterotoxins SEG and SEI.

With the present work a contribution can be given to a better understanding of the involvement of egc enterotoxins producing S. aureus strain in SFPOs. In addition, the results also give a first insight into the conditions and limits of egc enterotoxin production and a first indication in how they have the ability to be produced in food production and storage.

Figure 1 :

 1 Figure 1: From the outside inward: first three circles show distribution of genomic islands (GIs) (yellow=SCCmec; red=islands; pink=prophages), transposons (light blue=Tn5801; dark blue=Tn554), and insertion sequences (green) in the chromosomes of Mu50 (first circle), N315 (second circle), and MW2 (third circle). Red arrowheads indicate the attachment sites for GI families (*) and Sa1 (**). Third circle shows location of virulence genes cna (green arrow) and seh (red arrow). Fourth circle shows every 100th open reading frame (orf). Fifth and sixth circles show orfs on plus and minus strand, respectively. Blue=cell envelope and cellular processes; green=intermediary metabolism; orange=information pathways; yellow=other functions; magenta=similar to unknown proteins; pink=no similarity. Seventh and eighth circles show taxonomic distribution of BLAST best-hit entries on the plus and minus strand, respectively. Blue=Bacillus/Clostridium group; green=firmicutes (Gram-positive eubacteria); pink=viruses/insertion sequences/transposons; orange=archaea/eubacteria/eukaryota; white=no hit or ribosomal and transfer RNAs. Ninth circle shows virulence-associated orfs. Red=toxins; green=adhesins; orange=exoproteins; blue=others. Tenth circle shows orfs whose BLAST first hit entry is a human pathogen (red=streptococci; orange=Bacillus anthracis and Bacillus cereus; green=others). Eleventh circle shows GC content at the third codon (GC3) and synonymous codon-usage bias of each orf. Green=highly expressed orfs; red=putative alien orfs; orange=possible alien orfs based on GC3 skew; blue=other orfs. Size of the coloured bar=deviation of GC3 value of each orf from the average. Red arrowhead=rRNA and its orientation. Black bars=locations of tRNAs. Twelfth circle shows nucleotide position in Mb. (Baba et al., 2002)

Figure 2 :

 2 Figure 2: Acquisition of MGEs by S. aureus. 1 Incorporation of plasmids or plasmid elements into genomic DNA. 2 Plasmids can be maintained as free circular DNA. 3 Suicide plasmid. 4 Transfer of a transposon or an insertion sequence between plasmid and genomic DNA. 5 Transfer of a transposon or an insertion sequence between plasmids within the cell. 6 Transfer of a transposon or an insertion sequence from genomic DNA to another plasmid (Malachowa and DeLeo, 2010)

Figure 3 :

 3 Figure 3: Linear schematic of the USA300 genome (strain FPR3757) and its major MGEs (Malachowa and DeLeo, 2010).

Figure 4 :

 4 Figure 4: Representation of all Staphylococcus aureus genomic island vSaβ types I to XV, their virulenceassociated genes, and other hypothetical genes located on vSaβ. For each vSaβ type, one reference strain is shown. Arrows show orientation of open reading frames. Asterisks indicate truncated or fragmented genes. Note that vSaβ IV is substantially longer due to the presence of a complete phage. All other sequences are scaled relative to each other (Klaui et al., 2019).Plasmids

Figure 5 :

 5 Figure 5: Staphylococcal enterotoxin (SE) and SE-like toxin genes carried by Staphylococcus aureus pathogenicity islands, υSa genomic islands, prophages, and plasmids (Hu et al., 2018).

Figure 6 :

 6 Figure 6: Maximum likelihood phylogenetic tree of staphylococcal enterotoxins, including SEl26 and SEl27. The tree was constructed based on amino acid sequences using a Poisson model in MEGA 6.06. Bootstrap values (expressed as percentages of 1000 replications) are shown at the branch points. (Zhang et al., 2018)

  secondary structure is a mixture of α-helix and β-sheet components. Domain A contains both the amino and carboxyl termini, as well as a β-grasp motif. The amino-terminal residues drape over the edge of β-sheet in a loosely attached structure. The interfaces between A and B domains are marked by a set of α-helices, which form a long groove in the back side of the molecule and a shallow cavity at the top. The domain B is associated with binding to carbohydrates or nucleic acids. The internal β-barrel region is richly hydrophobic, and the external surface is covered by a number of hydrophilic residues. The characteristic SE disulphide bond is located at the end of domain B, opposing the α-helical cap. The resulting loop structure is flexible, although this seems to vary among the SEs, depending on the length of the loop[START_REF] Hu | Mechanisms of staphylococcal enterotoxin-induced emesis[END_REF].

Figure 7 :

 7 Figure 7: The complete amino acid sequence (A) and three dimensional structure (B) of SEA (Hu and Nakane, 2014).

Figure 8 :

 8 Figure 8: The biological multifunctionality of SEs in food poisoning, toxic shock and infection (Hu and Nakane, 2014)

Figure 9 :

 9 Figure 9: Diagram of normal antigen (A), superantigen binds to T cell receptor and MHC class II of APC (B), and the binding sites of SEA to these cells (C) (Hu and Nakane, 2014).

Figure 10 :

 10 Figure 10: The emetic response of house musk shrews against SEA administration. A small emetic animal model, house musk shrews (A, arrow indicates vomit) (Hu and Nakane, 2014).

Figure 11 :

 11 Figure 11: Proposed mechanism of enterotoxin-induced emesis. The enterotoxins transit through mucusexpelling goblet cells and epithelial cells in the intestinal epithelium to reach the lamina propria. Here, the enterotoxins can interact with mast cells to induce the release of 5-hydroxytryptamine (5-HT/serotonin precursor), which interacts with the vagus nerve to cause an emetic response. Additional cellular targets that may have possible roles in the induction of enterotoxigenic disease include different types of T cells and neutrophils (Fisher et al., 2018)

Figure 12 :

 12 Figure 12: Overview of Staphylococcal Food Poisoning (Fetsch and Johler, 2018)

Figure 13 :

 13 Figure 13: Number of food-borne and waterborne outbreaks in Europe, by causative agent (European Food Safety et al., 2019)

Figure 14 :

 14 Figure 14: Sankey diagrams of the distribution of food vehicles implicated in strong-evidence foodborne and waterborne outbreaks caused by bacterial toxins (N = 120), in the EU, 2018 (European Food Safety et al., 2019)

Figure 15 :

 15 Figure 15: Regulation of staphylococcal enterotoxins expression. Harsh bacterial growth conditions, changes in the bacterial microenvironment, high cell density, hypoxia, and membrane changes direct enterotoxin expression through the alternative σ factor, SarA protein family, Agr quorum sensing system, SrrAB protein, and SaeRS two-component system, respectively. The excitatory and inhibitory action of these systems on the other regulators and enterotoxins are summarized. Arrowheads represent upregulation and bars downregulation (Fisher et al., 2018)

Figure 16 :

 16 Figure 16: Schematic representation of the sea gene regulatory mechanism. Food parameters such as NaCl, weak acids, and preservatives may lead to prophage induction and replication of the circular, replicative form (RF) of the phage genome, resulting in an increase in RF copies in the cell. Prophage induction will initiate transcription from the latent promoter P2 resulting in the production of a longer sea transcript in addition to the sea transcript from the endogenous P1 promoter (Zeaki et al., 2019).

Figure 17 :

 17 Figure 17: Summary of real-time PCR response curves. A threshold level has to be set sufficiently high to differ from the background. The number of cycles required to reach threshold, Ct-or Cp-Value are assessed (Kubista et al., 2006)

Figure 18 :

 18 Figure 18: Summary of the processes in reverse transcriptase real time PCR (RT-qPCR). Modified according to www.sigmaaldrich.com

Figure 19 :

 19 Figure 19: Four different priming methods for the reverse transcription step in two-step assays of RT-qPCR (www.thermofisher.com).

Figure 20 :

 20 Figure 20: Absolute and relative quantification strategies in real-time RT-qPCR (Pfaffl et al., 2019).

Figure 21 :

 21 Figure 21: Overview of the study's aim



  ANSES: 143 SFPO strains isolated from the NRLs all over Europe  Agroscope (Mastitis): 153 strains collected in different studies from Hans-Ulrich Graber  Agroscope (Food): 161 strains from the study from Hummerjohann et al. (2014)  Strain collection of the Federal Food Safety and Veterinary Office (FSVO): 41 strains isolated from different foods (SFPO and non-SFPO)  Cantonal Laboratory Thurgau: 65 strains isolated from foods  Reference and NCBI strains: 9 strains, out of them two were available as isolates in the lab (Mu50, N315)



  Evaluation and prediction of staphylococcal enterotoxin expression (STEERS) Schwendimann. L.  IAFP Europe 2019 Nantes, April 2019 Expression and prediction of staphylococcal enterotoxin G and I Schwendimann, L., Merda, Berger, T., Hennekinne, J. A., Kläui, A. Nia, Y., Mistou, M.Y., Graber, H.U.  IAFP Europe 2020 Munich, April 2020 (cancelled) Behaviour of staphylococcal egc enterotoxins during bacterial growing and under food production-like stress conditions Schwendimann, L., Berger, T., Hennekinne, J. A., Ivanovic, I., Nia, Y., Merda, D. Mistou, M.Y., Graber, H.U. Poster presentation Effects of scalding temperature on the growth of Staphylococcus aureus and the formation of enterotoxins in the production of alpine hard cheese. In: International Scientific Conference on Raw Milk. 24 October, Valencia, Spain. 2019, 1.

  Six gènes (seg, sei, sem, sen, seo et seu), codant des entérotoxines supposées et indétectables, sont situés sur le locus (egc) qui fait partie de l'île génomique de Staphylococcus aureus vSaβ. Ces entérotoxines ont été décrites comme étant potentiellement impliquées dans des toxi-infections alimentaires collectives à staphylocoques. L'objectif de la présente étude était de déterminer si les données du génome complet peuvent être utilisées pour prévoir la production d'entérotoxines à staphylocoques egc, en particulier l'entérotoxine G (SEG) et l'entérotoxine I (SEI). Dans ce but, les séquences du génome entier de 75 souches de Staphylococcus aureus (S. aureus) d'origines différentes (intoxications alimentaires, humaines et animales) ont été étudiées en appliquant des méthodes bioinformatiques (analyse phylogénétique utilisant le génome de base et différents alignements). L'expression de SEG et de SEI a été testée in vitro à l'aide d'une méthode ELISA en sandwich. Les souches ont pu être affectées à 14 types vSaβ différents, chaque type étant associé à un complexe clonal individuel (CC). En outre, le type vSaβ et le CC ont été associés à l'origine de la souche (humaine ou bovine). La quantité de SEG et de SEI produite était également corrélée avec le type vSaβ et le CC d'une souche. Les résultats actuels fournissent des indications prometteuses permettant de supposer que la production in vitro de SEG et de SEI pourra être prédite sur la base du type vSaβ ou du CC d'une souche.Importance (French)Outre les infections qu'il peut causer chez l'homme et les animaux, Staphylococcus aureus peut produire différentes entérotoxines dans les aliments. Ces entérotoxines peuvent provoquer des vomissements et des diarrhées, qui touchent souvent de nombreuses personnes. La plupart de ces foyers passent encore inaperçus car les méthodes de détection des entérotoxines ne sont disponibles que pour quelques entérotoxines, mais pas pour les entérotoxines G (SEG) et I (SEI) découvertes plus récemment.Dans cette étude, nous avons obtenu des résultats prometteurs qui permettent de penser que la production in vitro de SEG et de SEI pourra être prédite à partir des données de séquençage du génome complet d'une souche. En outre, ces données pourraient être utilisées pour trouver la source (humaine ou bovine) d'une souche épidémique, ce qui permettrait de mieux comprendre le rôle de SEG et de SEI dans les épidémies d'origine alimentaire causées par S. can produce a variety of heat stable enterotoxins, which when they are secreted in food, can cause staphylococcal food poisoning outbreaks (SFPO). According to the European Food Safety Authority (EFSA) staphylococcal enterotoxins (SE) in mixed foods and meat products are among the top 10 pathogen/food vehicle pairs, causing the highest number of hospitalizations in strong evidence outbreaks. By looking at the number of cases, this trend seems to be on the rise (2019). In addition, most of the SFPO are classified as weak evidence outbreaks since only the so called classical enterotoxins (SEA, SEB, SEC, SED, and SEE)

Figure 22 :

 22 Figure 22: Representation of the newly defined S. aureus genomic island vSaβ types XVI to XXII. The virulence-associated genes, and other hypothetical genes located on vSaβ are also presented. For each vSaβ type, one reference strain is shown. Arrows show orientation of open reading frames. FIG number are hp genes which were assigned to a FIG number by the RAST (Rapid Annotations using Subsystem Technology) pipeline. Ent1 and ent2 of vSaβ type XVIII are genes that were already described in Colery & Smyth (Collery and Smyth, 2007). *Truncated or fragmented gene

Figure 23 :

 23 Figure 23: Maximum likelihood phylogenetic tree based on the core genome (nucleotidic sequences) showing the evolutionary relationship among 75 isolates of Staphylococcus aureus (all strains positive for the enterotoxin gene cluster) recovered from human, animal, environment and food samples (left). At the right, for each strain its clonal complex (CC), origin of the strain, source of the strain and their involvement in staphylococcal food poisoning outbreak (SFPO) is given. Bootstrap values are shown if >80. Production of enterotoxin G (SEG) and I (SEI) for the 32 analyzed strains is also given (last two columns), these are shown in "L" for low enterotoxin production and "H" for high enterotoxin production. * Statistical outliers **unknown CC

Figure 24 :

 24 Figure 24: Amount [ng/ml] of Staphylococcal enterotoxin G and I (component figure A and B, respectively), measured with Enzyme-linked immunosorbent assay. Each point is the average measurement from three biological replicates and the corresponding bars represent standard deviation. Strains were incubated in Brain heart infusion (BHI) for 24 h at 37 °C. The limit of detection of the corresponding enterotoxin is presented by -line (LOD SEG = 0.001 ng/ml, LOD SEI = 0.037 ng/ml).

  ), 77 des 114 foyers causés par les entérotoxines staphylococcociques (TIAC) sont difficiles à identifier. Cependant, seuls cinq des 25 entérotoxines staphylococciques peuvent être analysées à l'aide de kits disponibles dans le commerce. La présence de ce qu'on appelle les « nouvelles entérotoxines », en particulier, ne peut pas être identifiée. Un groupe de ces nouveaux gènes d'entérotoxines sont les gènes seg, sei, sem, sei, seo et seu qui sont situés sur le même locus (egc) et semblent être impliqués dans les TIAC.L'objectif de la présente étude est d'améliorer la compréhension des paramètres et des conditions dans lesquelles les entérotoxines egc sont produites. Ces connaissances permettront de mieux contrôler leur expression lors de la production et du stockage des aliments. Dans ce but, une sélection de huit souches d'origine différente a été choisie sur la base de leur diversité génétique (structure génétique) et de leur origine (humaine, animale, environnementale, alimentaire). Leur expression (ARNm) de seg, sei, sem, sen et seo a été mesurée par RT-qPCR à trois différents points de la phase de croissance de la bactérie (début, milieu et fin de la phase logarithmique). Sur base de ces résultats, trois souches ont été sélectionnées dans le but d'étudier l'expression de leurs entérotoxines dans des conditions de stress: concentrations de NaCl jusqu'à 100 g/l, température élevée (45 °C). En outre, chaque échantillon a été testé pour la recherche des entérotoxines staphylococciques G et I à l'aide d'une méthode interne ELISA en sandwich. Les résultats montrent que les entérotoxines egc sont principalement exprimées au milieu de la phase logarithmique de la croissance bactérienne et semblent s'éteindre à la fin de cette phase. SEG et SEI sont déjà produites à un stade précoce de la phase de croissance. Il est intéressant de noter que le stress lié au NaCl, qui imite les conditions de la production alimentaire, ne semble pas affecter l'expression et la production d'entérotoxines egc jusqu'à 20 g/l. Cependant, des différences ont été observées entre les souches.

Figure 25 :

 25 Figure 25: Schematic representation of the experimental set up for the different parameters tested and the sampling points.

Figure 26 :Figure 27 :

 2627 Figure 26: Average growth of S. aureus strains in BHI broth (in triplicates). Increase in cell density is measured at OD600. Samples were taken from the culture at the points plotted (from T0 toT4). The figure is presenting the growth of 8 S. aureus strains under standard conditions (37 °C, 5 g/L NaCl). * are presenting the adjusted sampling points, according to the individual behaviour of the strains

Figure 28 :

 28 Figure 28: Results of cultures of eight S. aureus strains collected at 4 time points of growth (T0: starting point, T1: mid-exponential; T2: late exponential; T3: stationary; and T4: late stationary growth phases) with 3 replicates (i.e. 3 independent cultures that were each sampled 4 times). Component Figure A is presenting the mean relative expression ratios (mRNA copies/cell) for seg, sei, sem, sen and seo (sampling points T0-T3). Gene expression levels were quantified using RT-qPCR. Normalization was provided using total RNA. Component Figure B is presenting the mean concentration ng/mL of enterotoxin G and I (sampling points T1-T4). Error bars indicate standard deviations.

Figure 29 :

 29 Figure 29: Results of cultures of three S. aureus strains under exposure to three different sodium chloride concentrations (12.5, 20, 100 g/L) with 3 replicates. Component Figure A is presenting the mean relative expression ratios at the mid-exponential growing phase (mRNA copies/cell) for seg, sei, sem, sen and seo (sampling points T1). Gene expression levels were quantified using RT-qPCR. Normalization was provided using total RNA. Component Figure B is presenting the mean concentration ng/mL of enterotoxin G and I at after 24h of incubation (sampling point T4).

Figure 31 :

 31 Figure 31: Characterisation, prediction and source tracking of an egc containing S. aureus strain potentially involved in a foodborne outbreakFirst step is taking an isolated S. aureus from the presumptive food matrix involved in a foodborne outbreak. Then it has to be decided if the strain is going to be directly sequenced or a series of qPCRs is going to be performed. Taking the first option has a lot of advantages as all genome information will be available sooner. On the other side the sequencing of the strain will cost more time and money.

  d'entérotoxines staphylococciques egc en se basant sur les données WGS. Cet objectif principal a pu être atteint et on a également pu trouver d'autres informations sur les entérotoxines egc et vSaβ. La corrélation entre le complexe clonal et le type de vSaβ, ainsi que le fait que la production d'entérotoxines G et I dépendait du type de vSaβ et qu'elle pouvait être statistiquement répartie entre production « élevée » et « faible », permet de prédire la production de SEG et de SEI sur la base des données du génome entier. En outre, les nouveaux résultats montrent également qu'en se basant sur le CC, on peut donner une indication sur la source possible de contamination. Ces nouvelles connaissances peuvent être utilisées dans le cas d'une TIAC à staphylocoques afin d'évaluer l'implication d'une certaine souche dans un foyer. Néanmoins, je pense que ce modèle de prédiction doit encore être étendu en raison de la diversité des complexes clonaux, afin de permettre la caractérisation génotypique et phénotypique des CC non encore décrits. Cela devrait être fait par l'analyse des vSaβ et le test de leur production d'entérotoxines G et I. En outre, dès qu'une méthode quantitative de détection des entérotoxines SEM, SEN et SEO sera disponible, les souches des différents types de vSaβ devraient être analysées pour la production de ces entérotoxines. Si les résultats donnent également les mêmes indications que ceux de SEG et SEI, le modèle pourrait être étendu à toutes les entérotoxines egc. La corrélation entre vSaβ et la production d'entérotoxines ayant été établie au départ, on a supposé que les différences étaient dues à la diversité des promoteurs. En raison de l'absence d'informations sur les TSS, les études sur les promoteurs n'ont pas pu être réalisées comme prévu. En conséquence, je pense qu'un ARNseq serait nécessaire pour pouvoir définir les TSS. En outre, cette méthode permettrait également de confirmer si les entérotoxines egc sont toutes exprimées ensemble ou séparément. En ce qui concerne la sécurité alimentaire, cette étude a également permis d'obtenir de nouvelles informations sur la production de SEG et de SEI selon différents paramètres. Dans le cadre des paramètres de stress, l'expression (ARNm) a été mesurée pour toutes les entérotoxines egc. Néanmoins, il n'est pas possible de se prononcer sur les protéines pour SEM, SEN et SEO, car nous ne savons pas si la traduction de l'ARNm en protéines s'est produite pour ces entérotoxines. Il est surprenant de constater que la production de SEG et de SEI de ces entérotoxines a pu être mesurée deux heures déjà après l'inoculation, avec une température d'incubation de 45°C ou une concentration de NaCl allant jusqu'à 100 g/l. Ces résultats montrent que SEG et SEI peuvent également être produites dans des conditions peu favorables. Par conséquent, il convient d'observer plus en détail comment elles sont produites dans une matrice alimentaire. En particulier pour les aliments qui nécessitent beaucoup de manipulation humaine (fabrication artisanale de fromage et autres), des tests de provocation doivent être effectués afin de déterminer si ces entérotoxines peuvent être produites dans des conditions réelles. L'enterotoxin gene cluster egc est composé de cinq gènes d'entérotoxine pour lesquels une activité émétique est démontrée. À mon avis, ce cluster devrait être considéré comme une seule et même entité et, à l'avenir, si l'on dispose de plus de connaissances sur les entérotoxines individuelles et leur comportement. Peut-être qu'une seule entérotoxine représentative (une sur cinq) pourra être utilisée pour démontrer l'implication d'une souche de S. aureus positive au gène egc dans une TIAC à staphylocoques. Néanmoins, avant d'en arriver là, une méthode de détection devrait être disponible pour les cinq entérotoxines afin de pouvoir étudier leur mode de production de toxines et de savoir si la quantité totale d'entérotoxines produites peut être dangereuse dans les cas de toxi-infections alimentaires.

Figure 1 : 12 Figure 2 : 13 Figure 3 : 14 Figure 4 :

 1122133144 Figure 1: From the outside inward: first three circles show distribution of genomic islands (GIs)

Figure 5 : 23 Figure 6 : 25 Figure 7 : 26 Figure 8 : 27 Figure 9 :

 5236257268279 Figure 5: Staphylococcal enterotoxin (SE) and SE-like toxin genes carried by Staphylococcus

Figure 10 :Figure 11 :Figure 12 :Figure 13 :Figure 14 :Figure 15 :Figure 16 :Figure 17 :Figure 18 :Figure 19 :Figure 20 :Figure 21 :Figure 22 :Figure 23 :Figure 24 :Figure 25 :Figure 26 :Figure 27 :Figure 28 :Figure 29 :Figure 30 :

 101112131415161718192021222324252627282930 Figure 10: The emetic response of house musk shrews against SEA administration. A small emetic animal model, house musk shrews (A, arrow indicates vomit) (Hu and Nakane, 2014). ..........................................................................................................................31

Figure 1 .

 1 Figure 1. Flow chart for the production of cheese in the laboratory cheese-making model. The white boxes represent the processes applied, and the light grey boxes provide information on material added/removed during the cheese making process. The dark grey boxes refer to sample collection at different times in the cheese making process. *The heating and holding rates are described in Table2.

Figure 1

 1 Figure 1 provides an outline of the experiments. An appropriate volume of thawed inoculum (calculated based on the concentration of the frozen inoculum, reaching a concentration in the milk of ~10 4 CFU/ml) and 100 μl of Calcium chloride (CaCl2, 35%) were added to each bottle containing 200 ml of raw milk. Using a thermostat, the mixture was then heated to 30-32° C, with stirring. Thereafter, 200 μl of thermophilic starter RMK 150 (Agroscope, Bern, Switzerland)

Figure 2 .

 2 Figure 2. Concentration (CFU/g) with standard deviation of S. aureus strains during the cheese making process for samples raw milk (with inoculum) [A], curd [S], and cheese [K20] (see also Fig. 1). Component Figures 2A-2E correspond to strains a-e respectively. The different markers denote the different scalding temperatures applied (46-56° C)

  Staphylococcus aureus, staphylococcal enterotoxins, enterotoxin gene cluster (egc), SEG, SEI, prediction, stress parameters Abstract :

Table 1 : Factor accepting growth of Staphylococcus aureus (Hennekinne et al., 2012; Tatini, 1973)

 1

		Optimum	Range
	Temperature [°C]	37	7-48
	pH	6-7	4-10
	Water activity [aW]	0.98	0.83 → 0.99*
	NaCl [%]	0	0-20
	Redox potential [Eh]	> +200mV	< -200 mV to > +200 mV
	Atmosphere	Aerobic	Anaerobic-aerobic

Table 2 : Overview of staphylococcal enterotoxins

 2

	Toxin	Molecular weight [kDa]	Gene localisation	Reference
	SEA	27.1	Prophage	(Betley and Mekalanos, 1985)
	SEB	28.4	Plasmid/pathogenicity island	(Shafer and Iandolo, 1978)
	SEC	27.5	Plasmid/pathogenicity island	(Bohach and Schlievert, 1987)
	SED	26.9	Plasmid	(Bayles and Iandolo, 1989)
	SEE	26.4	Prophage	(Couch et al., 1988)
	SEG	27	Prophage (egc)	(Jarraud et al., 2001)
	SEH	25.1	Transposom	(Ren et al., 1994)
	SEI	25.1	Prophage (egc)	(Jarraud et al., 2001)
	SElJ	28.2	Plasmid	(Zhang et al., 1998)
	SElK	25.4	Prophage	(Orwin et al., 2001)
	SEL	24.7	Plasmid/pathogenicity island	(Fitzgerald et al., 2001)
	SEM	24.8	Prophage (egc)	(Jarraud et al., 2001)
	SEN	26.1	Prophage (egc)	(Jarraud et al., 2001)
	SEO	26.8	Prophage (egc)	(Jarraud et al., 2001)
	SEP	26.7	Prophage	(Omoe, Imanishi, et al., 2005)
	SEQ	25.1	Prophage	(Hu et al., 2017)
	SER	27	Plasmid	(Omoe et al., 2003)
	SES	26.2	Plasmid	(Ono et al., 2008)
	SET	22.6	Plasmid	(Ono et al., 2008)
	SElU	28.6	Prophage (egc)	(Thomas et al., 2006)
	SElV	23.2	Prophage (egc)	(Thomas et al., 2006)
	SElX	23.3	Chromosome	(Langley et al., 2017)
	SElY	22.5	-	(Ono et al., 2015)
	SElZ	-	-	(Spoor et al., 2015)
	SEl26	25.08	Prophage	(Zhang et al., 2018)
	SEI27	26.9	Prophage	(Zhang et al., 2018)

Table 3 : Emetic and superantigenic activity of Staphylococcal enterotoxin and Staphylococcal enterotoxin like. NE not examined, ED50 = 50% emetic dose (according to Hu et al. 2018) SEs/SEl Superantigenic activity Emetic activity [μg/animal] Monkey 1 House Musk Shrew 2

 3

	SEA	+	5 (ED50)	0.3
	SEB	+	5 (ED50)	10
	SEC	+	5 (ED50)	1000
	SED	+	5 (ED50)	40
	SEE	+	10-20 (ED50)	10
	SEG	+	160-320	200
	SEH	+	30	1000
	SEI	+	300-600	1
	SElJ	+	NE	NE
	SElK	+	100 (2/6)	NE
	SEL	+	100 (1/6)	NE
	SEM	+	100 (1/7)	NE
	SEN	+	100 (1/8)	NE
	SEO	+	100 (2/8)	NE
	SEP	+	100 (3/6)	50
	SEQ	+	100 (2/6)	NE
	SER	+	<100	<1000
	SES	+	<100	20
	SET	+	<100	1000
	SElU	+	NE	NE
	SElV	+	NE	NE
	SElX	+	NE	NE
	SElY	+	NE	500
	SEIZ	+	NE	NE
	SEl26	+	NE	NE
	SEI27	+	NE	NE
	1 Oral administration
	2 Intraperitoneal administration

Table 4 : Distribution of Staphylococcal enterotoxin types for Staphylococcus aureus strains isolated from food-poisoning cases and different foods and food-handlers 1 (Chiang et al., 2008), 2 (Merda et al., 2020), 3 (Sato'o et al., 2014), 4 (Argudin et al., 2012), 5 (Hummerjohann et al., 2014) NA = not analyzed Description SFPO Taiwan (147 isolates) 1 SFPO Europe (143 isolates) 2 SFPO Japan (42 isolates) 3 Food and - handlers Spain (64 isolates) 4 Cheese Switzerland (102 isolates) 5 Se gene incidence [%] in S. aureus isolates

 4

	sea	29.20	34.97	70.00	38.70	21.00
	seb	19.70	6.29	45.00	12.90	NA
	sec	6.80	14.69	7.00	16.10	1.00
	sed	2.00	11.89	5.00	22.60	44.00
	see	-	2.80	-	-	NA
	seg	2.00	34.97	20.00	80.70	14.00
	seh	8.20	17.48	55.00	9.70	4.00
	sei	29.90	37.76	20.00	8.70	16.00
	seij	2.00	13.99	5.00	22.60	42.00
	seik	16.30	13.99	55.00	6.50	NA
	sel	6.80	13.99	3.00	9.70	NA
	sem	11.60	35.66	17.00	80.70	NA
	sen	10.90	37.76	17.00	80.70	NA
	seo	14.30	37.06	17.00	80.70	NA
	sep	27.90	11.89	10.00	9.70	1.00
	seq	10.90	14.69	55.00	9.70	NA
	ser	5.40	13.99	5.00	16.10	42.00
	ses	NA	0.70	NA	-	NA
	set	NA	0.70	NA	-	NA
	seiu	14.20	25.17	NA	48.40	NA
	seiv	NA	0.00	NA	NA	NA
	seix	NA	12.59	NA	NA	NA
	seiy	NA	89.51	NA	NA	NA
	seiz	NA	9.09	NA	NA	NA
	sei26	NA	0.00	NA	NA	NA
	sei27	NA	0.00	NA	NA	NA
	From the data of Table 4 it can be observed that not only classical enterotoxins (sea-see) are
	harboured by S. aureus. From the classical ones, sea seems to be the most prevalent one (21-
	70%) and see the less prevalent one (0-2.8%). From the non-classical ones, sey seems, to be
	the most prevalent one, as this enterotoxin is one of the newer ones, not much studies are

Table 5 : Epidemiological data, coagulase positive staphylococci (CPS) concentrations, staphylococcal enterotoxin genes (se) and staphylococcal toxin (SE) detected from different staphylococcal food poisoning outbreaks reported in literature. * VIDAS set2 results (detecting SEA-SEE) Country Product Cases CPS [CFU/ml] Se genes SE confirmed Reference

 5

	Romania	Sheep curd cheese	8	10 6 -10 8	sed, sej,	SEA-SEE*	(Ciupescu et al.,
					ser		2018)
	Romania	Appetizer made with	52	10 6	sed, sej,	SEA-SEE*	(Ciupescu et al.,
		soft sheep cheese			ser, egc		2018)
	Romania	Raw cow milk ripened	36	10 6	seh	Negative*	(Ciupescu et al.,
		salted cheese					2018)
	Switzerland	Tomme soft cheese	14	10 7	sea, sed,	SEA, SED	(Johler, Weder, et
					sej, ser,		al., 2015)
					egc
	Switzerland	Goat fresh cheese	5	10 6 -10 7	egc	Negative*	(Johler, Giannini,
							et al., 2015)
	Switzerland	Goat semi-hard cheese 6	10 3	egc	Negative*	(Johler, Giannini,
							et al., 2015)
	Japan	powdered skim milk	13420 not	-	SEA	(Asao et al., 2003)
				detected
	Japan	Skim milk powder	10000 -	sea, seh,	SEA, SEH	(Ikeda et al.,
					egc		2005)
	Norway	Mashed potato with raw	8	10 8	seh	SEH	(Jorgensen et al.,
		milk					2005)
	Japan	Sushi, potato salad,	15	10 4 -10 8	egc	Negative*	(Umeda et al.,
		fried barbecue/shrimp/					2017)
		chicken, sandwich, raw
		vegetable
	Belgium	Mashed potato	28	10 2	sea, sed,	SEA, SED	(Denayer et al.,
					sej, ser,		2017)
					egc
	Belgium	Chicken, sausage,	18	10 6	sea, sec	SEA, SEC	(Denayer et al.,
		bovine meat, potato					2017)
		preparation
	Belgium	Mashed potatoes with	6	10 7	sea, egc	SEA	(Denayer et al.,
		carrots					2017)
	Italy	Chantilly Cream	24	10 8	sea	SEA	(Ercoli et al.,
		Dessert					2017)

Table 6 : S. aureus enterotoxins (SEB, SEC SED) regulated by Agr, SarA, σB, Rot, and SaeRS modified from (Zeaki et al., 2019).

 6

	Enterotoxins	Agr	SarA	σB	Rot	SaeRS	Reference
	SEB	+/0	+	-	-	+

Table 7 : Factors affecting enterotoxin production of Staphylococcus aureus (Hennekinne et al., 2012; Tatini, 1973)

 7

		Optimum	Range
	Temperature [°C]	37-45	10-45
	pH	7-8	4-9.6
	Water activity [aW]	0.98	0.85 → 0.99
	NaCl [%]	0	0-10
	Redox potential [Eh]	> +200mV	< -100 mV to > +200 mV
	Atmosphere	Aerobic (5-20% dissolved O2)	Anaerobic-aerobic

Table 8 : Process hygiene and food safety criteria according to commission regulation (EC) No 1441/2007 and HyV 2016

 8

	Food category	Bacteria/toxin	Sampling plan	Limits		Analytical method	Stage were criteria applies	Action in case of unsatisfactory results
			n	c	m	M
	Cheeses, milk powder and whey powder	enterotoxins Staphylococcal	5	0	25 g Not detected in	1 or 2 SN EN ISO 6888-	shelf-life Products placed on the market during their	-
		Coagulase-
	Cheeses made from raw milk	positive staphylococci	5	2 10 4 cfu/g 10 5 cfu/g EN/ISO 6888-2	At the time during the manufacturing
								process when the	Improvements in
								number of	production hygiene
	Cheeses made from milk that has							staphylococci is	and selection of raw
	undergone a lower heat treatment than pasteurisation and ripened cheeses made from milk or whey that has undergone pasteurisation or a stronger	Coagulase-positive staphylococci	5	2	100 cfu/g	1 000 cfu/g	EN/ISO 6888-1 or 2	expected to be highest	materials. If values > 10 5 cfu/g are detected, the cheese batch has to be
	heat treatment								tested for
	Unripened cheeses) made from milk or whey that soft cheeses (fresh has undergone pasteurisation or a stronger heat treatment	Coagulase-positive staphylococci	5	2 10 cfu/g	100 cfu/g	EN/ISO 6888-1 or 2	End of the manufacturing process	staphylococcal enterotoxins.
	Milk powder and whey powder	Coagulase-positive staphylococci	5	2 10 cfu/g	100 cfu/g	EN/ISO 6888-1 or 2	End of the manufacturing process
	Shelled and shucked products of cooked crustaceans and molluscan shellfish	Coagulase-positive staphylococci	5	2	100 cfu/g	1 000 cfu/g	EN/ISO 6888-1 or 2	End of the manufacturing process	Improvements in production hygiene

CPS = Coagulase positive staphylococci; SEs = Staphylococcal enterotoxins; n = number of units comprising the sample; c = maximum allowable number of sample units with values between m and M

Table 9 : Summary of commonly used Whole Genome Sequencing platforms (Jagadeesan et al., 2019).

 9

							Type of
		Sequencing			Error		instrument and
	Plattform	Technology Read length Output/run	rate	Example of use	run time
	Illumina	Sequencing by synthesis	Short reads × 300bp 1 × 36bp -2	0.3-1000Gb Low	Variant calling	Benchtop 2-29 h
	Ion Torrent	Sequencing by synthesis	Short reads 200-400bp	0.6-15Gb	Low	Variant calling	Benchtop 2-4 h
	PacBio	Single molecule sequencing by synthesis	Long reads Up to 60kb	0.5-10Gb	High	De novo finishing assembly of small and large genome bacterial genomes	Large scale 0.5-4 h
	Oxford Nanopore	Single molecule	Long reads up to 100kb	0.1-20Gb	High	Complete genome metagenomics of isolates and	Portable 1min-48 h

   16S-23S rRNA intergenic spacer PCR (RS-PCR): Rapid and fast method for genotyping of S. aureus strains, described inFournier et al. (2008)  DNA isolation and preparation for WGS: internal protocol already largely implemented for DNA isolation of S. aureus. Publication of this method, with the publication of the data from this study.La recherche bibliographique a permis de définir les objectifs de recherche et le type d'entérotoxines à étudier. Sur la base de ces résultats, ce sont les entérotoxines egc (seg, sei, sem, sen, seo et seu) qui ont été choisies. Cette décision a été prise en fonction des faits

	Objectifs de recherche (French) 2. Sélection d'un sous-ensemble de ces souches et, si les données ne sont pas 4 Valorisation
	disponibles, séquençage par WGS.
	Peer reviewed publications:
	3. Application de méthodes bioinformatiques pour créer un allignment de génome
	permettant de localiser les vSaβ et leur disposition dans le génome.
	4. Analyse des régions de promoteur d'entérotoxines egc des différents vSaβ  Schwendimann, L.; Berger, T.; Graber, H.U.; Meier, S.; Hummerjohann, J.; Jakob, E.
	5. Sur la base de la diversité du type vSaβ, de la diversité des origines et de la diversité (2020). Growth of Staphylococcus aureus, staphylococcal enterotoxin formation, and
	génétique, un sous-ensemble de souches est sélectionné et analysé pour leur the effect of scalding temperature during the production of Alpine cheese in a laboratory
	production de SEG et de SEI cheese-making model. J Food Prot, 83 (10): 1822-1828. 10.4315/JFP-19-600.
	9. Développement d'un modèle prédictif
	spa-type
	Different methods were already implemented in the respective labs,
	 vSaβ typing: method described by Klaui et al. (2019) Pour atteindre cet objectif, le travail a été divisé en différentes tâches:
	 ELISA method for detection of SEG and SEI: Development of these methods by CEA 1. Recherche d'une collection de souches disponibles contenant des souches de S.
	and implementation at Anses laboratory. Validation published in Feraudet Tarisse et aureus provenant d'aliments, de TIAC, de l'environnement et d'animaux.
	al. (2021) Caractérisation pour leur présence d'egc

Different origin: country/continent  Different source of isolation: Human, animal, environment, SFPO  Different results in subtyping by applying following methods: Sequence type, genotype, suivants:  Les entérotoxines egc semblent être présentes dans ~50 % des souches de S. aureus  L'activité émétique de seg, sei, sem et seo a été démontrée  On sait peu de choses sur l'expression des entérotoxines egc, sur la manière dont elles sont régulées et dont les différents paramètres de stress influencent leur expression  Des études récentes semblent montrer l'implication des entérotoxines egc dans les toxi-infections alimentaires Avec le choix des entérotoxines, l'objectif de cette étude est de trouver un modèle permettant de prédire la production d'entérotoxines egc sur la base des données du WGS, telles que présentées dans Figure 21. Plus en détail, le vSaβ (contenant egc) d'une grande collection de souches est analysé insilico. L'expression des systèmes de gènes se est ensuite étudiée par PCR quantitative à transcription inverse (RT-qPCR) en utilisant différents facteurs de stress (conditions environnementales). Ces résultats sont ensuite confirmés et comparés aux résultats obtenus avec le test ELISA (pour la détection de SEG et SEI) dans le but final de prédire l'expression de SE sur la base d'une analyse in-silico des données WGS. 6. Une méthode est développée (RT-qPCR) afin de mesurer l'expression des entérotoxines egc (seg, sei, sem, sen, seo) 7. Sur la base du résultat du point 5, différentes souches sont sélectionnées et la corrélation entre vSaβ et l'expression de se est étudiée à l'aide de la méthode RT-qPCR. En outre, l'expression génétique des entérotoxines egc sera évaluée sous différents paramètres de stress tels que le teneur en NaCl et la température. 8. Les résultats obtenus au point 6 sont comparés aux résultats obtenus à l'aide des méthodes ELISA pour la détection du SEG et du SEI.  Schwendimann, L.; Mérda, D., Berger, T.; Denayer, S.; Feraudet Tarisse, C.; Kläui, A.; Messio, S.; Mistou, M.Y.; Nia, Y.; Hennekinne, J.A., Graber, H.U. (2021). Staphylococcal enterotoxin gene cluster: prediction of enterotoxin (SEG and SEI) production and of the source of food poisoning based on vSaβ typing. Appl Env Micr, 87(5). 10.1128/AEM.02662-20.

Table 10 : Genotypic characteristics (i.e. clonal complex, enterotoxin genes present on the genome, vSaβ type, and spa-type) and origins of the 75 studied strains.

 10

	Strain	Country	Origin	Source of isolation		CC	Enterotoxin genes	vSaβ type	Spa-type
	07CEB94STA	Belgium	Food (SFPO)	Ready to eat	5	a, g, i, m, n, o. x	I	t704
	11CEB145STA	Japan	Human	Infection		5	a, c, g, i, m, n, o, x	I	*
	13CEB178STA	Ireland	SFPO	NA		5	d, j, g, i, m, n, o, r,x	I	t463
	13CEB188STA	Ireland	Food (SFPO)	Milk product	5	g, i, m, n, o, x	I	t5829
	13CEB191STA	Ireland	Food (SFPO)	Milk product	5	d, g, i, j, m, n, o, r, x	I	t837
	13CEB329STA	Belgium	Human (SFPO)	Nose throat	and	5	g, i, m, n, o, x	I	t7506
	15SBCL1507STA Ireland	Food (SFPO)	Meat		5	g, i, m, n, o, x	I	*
	15SBCL1550STA Ireland	Food (SFPO)	Ready to eat	5	g, i, m, n, o, x	I	t450
	17SBCL08STA	France	Food (SFPO)	Meat		5	g, i, m, n, o, x	I	t111
	17SBCL09STA	France	Food (SFPO)	Meat		5	g, i, m, n, o, x	I	t586
	17SBCL580STA	Bulgaria	Food (SFPO)	Ready to eat	5	a, d, g, i, j, m, n, o, r, x	I	t535
	17SBCL585STA	Bulgaria	Food (SFPO)	Ready to eat	5	a, d, g, i, j, m, n, o, r, x	I	t535
	502A	USA	Human	Infection		5	g, i, m, n, o	I	t010
	Mu50	Japan	Human	Infection		5	a, c, g, i, l, m, n, o, tst, x	I	t002
	N315	Japan	Human	Human faeces	5	c, g, i, l, m, n, o, p, tst, x	I	t002
	NZAK3	New Zealand	Human	Skin		5	c, g, i, l, m, n, o, p, x	I	t002
	ST288	England	Human	Urine		5	g, i, m, n, o	I	t1003
	18SBCL679	Switzerland	Food (SFPO)	Milk product	9	g, i, m, n, o, u, x, y, 27	XIII	t899
	G19F	Italy	Animal	Mastitis (cow)	9	g, i, m, n, o, u	XIII	t100
	13CEB177STA	Ireland	NA	FPO		10	c, g, i, m, n, o, u, x	XVII	t148
	11CEB277STA	Italy	Food	Milk product	20	g, i, m, n, o, u, x	XII	t3929
	11CEB279STA	Italy	environment	NA		20	g, i, m, n, o, u, x, y	XII	t325
	15SBCL1292STA France	Food (SFPO)	Milk product	20	g, i, m, n, o, u, x	XII	*
	15SBCL1299STA France	Food (SFPO)	Ready to eat	20	g, i, m, n, o, u. x. y	XII	t164
	15SBCL1397STA France	Food (SFPO)	Milk product	20	g, i, m, n, o, tst, u, x	XII	t164
	15SBCL1409STA France	Food (SFPO)	Milk product	20	g, i, m, n, o, u, x, y	XII	*
	15SBCL1428STA France	Food (SFPO)	Milk product	20	g, i, m, n, o, u, x, y	XII	*
	17SBCL202STA	France	Food (SFPO)	Milk product	20	g, i, m, n, o, u, x, y	XII	t164
	17SBCL208STA	France	Food (SFPO)	Milk product	20	g, i, m, n, o, u, x, y	XII	t458
	17SBCL214STA	France	Food (SFPO)	Milk product	20	g, i, m, n, o, u, x, y	XII	*
	17SBCL220STA	France	Food (SFPO)	Milk product	20	g, i, m, n, o, u, x, y	XII	t10134
	17SBCL225STA	France	Food (SFPO)	Milk product	20	g, i, m, n, o, u, x, y	XII	*
	18 SBCL 680	Switzerland	Food	Milk product	20	g, i, m, n, o, u, x, y	XII	t1544
	18 SBCL667	Switzerland	Food	Milk product	20	g, i, m, n, o, u, x, y	XII	*
	G11F	Switzerland	Animal	Mastitis (cow)	20	g, i, m, n, o, u	XII	t2736
	13CEB179STA	Ireland	NA	FPO		22	c, g, i, m, n, o, u, x	XVI	*
	15SBCL1517STA Ireland	Food (SFPO)	Meat		22	c, g, i, l, m, n, o, u, x	XVI	t645
	15SBCL1527STA Ireland	Food (SFPO)	Ready to eat	22	g, i, m, n, o, u, x	XVI	*
	13CEB181STA	Ireland	Food (SFPO)	Ready to eat	30	a, g, i, m, n, o, u	III	t3018

Table 11 : Ammino acid similarity of SEG and SEI [%] compared to those of the reference strains (Mu50, vSaβ type I). Each vSaβ type sequence is represented based on 100 % inter-group similarity.

 11

	vSaβ type	SEG	SEI
	I	100% R	100% R
	III	97%	95%
	IV	*	95%
	XI	97%	93%
	XII	100%	100%
	XIII	99%	100%
	XVI	100%	99%
	XVII	96%	97%
	XVIII	100%	100%
	XIX	97%	93%
	XX	99%	100%
	XXI	*	97%
	XXII	100%	99%
	R Reference
	* gene absent

Table 12 : Primers for detection of enterotoxin genes developped and used in this study. S for sense primers, AS for antisense primers Gene Primer Sequence 5'-3' Amplicon size (bp)

 12

	sem	Gsem_S	GATGTCGGAGTTTTGAATCTTA	584
		Gsem_AS	ACTTTCAGCTTGCCCTGTT
	sen	Gsen_S	TTCTTCCAGTTAAGCCTACACA	218
		Gsen_AS	CTGATATAACGTGGCAATTAG
	seo	Gseo_S	TAAAGCGCATTGTCATGGTGAG	348
		Gseo_AS	ACATCACTAGTCATTCGGTCATA

Table 13 : Strains used in this study and their characteristics

 13

	Strain name	Abbreviation	vSaβ type	CC Country	Origin	SFPO
	Mu50	Mu50, M	I	5	Japan	Human	no
	18SBCL669	26.3	XI	479 Switzerland	Milk product	no
	13CEB323	13ceb	XX	72	Belgium	Human	yes
	18SBCL673	KS24, K	XXII	45	Switzerland	Milk product	yes
	15SBCL1397	15SB, S	XII	20	France	Milk product	yes
	18SBCL670	26.1	IV	705 Switzerland	Milk product	no
	13CEB327	13	III	30	Belgium	Human	yes
	18SBCL672	AG	XXI	50	Switzerland	Milk product	No

Table 14 : Primers and Probes used in this study for standard PCR and real-time PCR

 14

	Target	Primer/ probe	Sequence 5'-3'	Amplicon size (bp)	Reference
	Standard
	PCR
	seg	Gseg-S	ACC TGA ACT ATC TAT AAG CGT GAA T	644	Boss et al. 2011
		Gseg-AS TCT TTA GTG AGC CAG TGT CTT G
	sei	Gsei-S	TAT AGA TTT AAA AGG CGT CAC	444	Cosandey et al. 2016
		Gsei-AS	ACT TTC CCI TTA TTA AAA CCT
	sem	Gsem-S	CGC TGA TGT CGG AGT TTT GAA TCT TA	576	This study
		Gsem-AS AAG AAA CTT TCA GCT TGT CCT GTT
	sen	Gsen-S	CTG ATA TAA CGT GGC AAT TAG	525	This study
		Gsen-AS GCT CCC ACT GAA CCT TTT AC
	seo	Gseo-S	ACT AAT CGC TTT ACA ATA AAT GAG TC 476	This study
		Gseo-AS TCC GAA TGC GAA TGG AAT TTA ATA
	nuc	Nuc-S	CTG GCA TAT GTA TGG CAA TTG TT	664	Boss et al. 2011
		Nuc-AS	TAT TGA CCT GAA TCA GCG TTG TCT
	Real-time
	PCR
	seg	qGseg-S	AGA CAT TTT TGG CGT TCC ATA	106	This study
		qGseg-AS	ATT TTG GTT TAT ATC CGG TTC AG
	sei	qGsei-S	AAC TGG ATA TTT TTG GCA TTG	116	This study
		qGsei-AS AAG ATT AAT AGG GAT TTT TCT AGC A
	sem	qGsem-S CTG ATG TCG GAG TTT TGA ATC TTA	60	This study
		qGsem-AS	GGT GGT CTT CAA TTG GAT AGC TAC
	sen	qGsen-S	CTT ATA CGG AGG AGT TAC GAT ACA T	68	This study
		qGsen-AS	CAT TAA CGC CTA TAA CTT TCT CTT C
	seo	qGseo-S	ATG ACC GAA TGA CTA GTG ATG	54	This study
		qGseo-AS	GAA TGC GAA TGG AAT TTA ATA
	nuc	qNuc-S	CCTGAAGCAAGTGCATTTACGA	166	Graber et al. 2007
		qNuc-AS	CTTTAGCCAAGCCTTGACGAACT
			CATCAGCATAAATATACGCTAAGC
		qNuc-P	CACGTCCA*
	*Minor groove binder qPCR probe labeled with FAM at the 5′ end and with a confidential nonfluorescent
	quencher at the 3′ end.

  Zhao, Y., Zhu, A., Tang, J., Tang, C., Chen, J., 2017b. Identification and measurement of staphylococcal enterotoxin M from Staphylococcus aureus isolate associated with staphylococcal food poisoning. Lett Appl Microbiol 65, 27-34, 10.1111/lam.12751. Zhu, W., Clark, N.C., McDougal, L.K., Hageman, J., McDonald, L.C., Patel, J.B., 2008.

	Vancomycin-resistant Staphylococcus aureus isolates associated with Inc18-like vanA
	plasmids in Michigan. Antimicrob Agents Chemother 52, 452-457, 10.1128/AAC.00908-
	07.

Table 1 :

 1 Factor accepting growth of Staphylococcus aureus(Hennekinne et al., 2012;[START_REF] Tatini | Influence of food environments on growth of Staphylococcus aureus and production of various enterotoxins[END_REF] ...........................................................................................................................10

Table 2 :

 2 Overview of staphylococcal enterotoxins ................................................................22

Table 3 :

 3 Emetic and superantigenic activity of Staphylococcal enterotoxin and Staphylococcal enterotoxin like. NE not examined, ED50 = 50% emetic dose (according to Hu et al. 2018) ....................................................................................................................................28

Table 4 :

 4 Distribution of Staphylococcal enterotoxin types for Staphylococcus aureus strains isolated from food-poisoning cases and different foods and food-handlers 1 (Chiang et al., 2008), 2 (Merda et al., 2020), 3 (Sato'o et al., 2014), 4 (Argudin et al., 2012), Hummerjohann et al., 2014) NA = not analyzed ........................................................33

5 (

Table 5 :

 5 Epidemiological data, coagulase positive staphylococci (CPS) concentrations, SEE) .......................................................................................39

	staphylococcal enterotoxin genes (se) and staphylococcal toxin (SE) detected from
	different staphylococcal food poisoning outbreaks reported in literature. * VIDAS set2
	results (detecting SEA-

Table 6 :

 6 S. aureus enterotoxins (SEB, SEC SED) regulated by Agr, SarA, σB, Rot, and SaeRS modified from[START_REF] Zeaki | The Role of Regulatory Mechanisms and Environmental Parameters in Staphylococcal Food Poisoning and Resulting Challenges to Risk Assessment[END_REF]. ...............................................................................44

Table 7 :

 7 Factors affecting enterotoxin production of Staphylococcus aureus(Hennekinne et al., 2012;[START_REF] Tatini | Influence of food environments on growth of Staphylococcus aureus and production of various enterotoxins[END_REF] .................................................................................................46

Table 8 :

 8 Process hygiene and food safety criteria according to commission regulation (EC) No 1441/2007 and HyV 2016 ............................................................................................51

Table 9 :

 9 Summary of commonly used Whole Genome Sequencing platforms (Jagadeesan et al., 2019). ....................................................................................................................65

Table 10 :

 10 Genotypic characteristics (i.e. clonal complex, enterotoxin genes present on the genome, vSaβ type, and spa-type) and origins of the 75 studied strains. ....................85

Table 11 :

 11 Ammino acid similarity of SEG and SEI [%] compared to those of the reference strains (Mu50, vSaβ type I). Each vSaβ type sequence is represented based on 100 % inter-group similarity. ...................................................................................................89

Table 12 :

 12 Primers for detection of enterotoxin genes developped and used in this study. S for sense primers, AS for antisense primers .....................................................................99

Table 13 :

 13 Strains used in this study and their characteristics .............................................. 112

Table 14 :

 14 Primers and Probes used in this study for standard PCR and real-time PCR ...... 116

  and higher temperatures no enterotoxin production takes place. All hard and extra-hard Swiss cheese production includes a process step in which the scalding temperature exceeds 52° C. Thus, provisional best practice guidelines for the manufacture of hard and extra-hard Swiss Alpine cheese[START_REF] Jakob | Leitlinie für die gute Verfahrenspraxis bei der Milchgewinnung und -verarbeitung in Sömmerungsbetrieben [Guideline for good practice in milk production and processing in summer mountain farms[END_REF] do not include a requirement for staphylococcal and SE tests of these cheeses. The objective of this study was to provide scientific data on microbiological safety of hard and extra-hard cheeses and to check the influence of the process and the scalding temperatures applied on staphylococcal enterotoxin formation. The Federal Office for Food Safety and Veterinary Affairs, as the responsible authority, has questioned this and commissioned the National Reference Laboratory for coagulase positive staphylococci

5

CFU/g at this time, SE tests of the lot must be conducted.

Based on practical experience of the advisory service for cheesemakers, it has been assumed that a scalding temperature of 52° C is sufficient to cause inactivation of all CPS present, and VI for this (Agroscope).

Table 1 : Description of the S. aureus strains (a-e) used in this study. Data taken from Hummerjohann et al. (2014). SET, staphylococcal enterotoxin; RPLA, detection by reversed passive latex agglutination; spa- type, technique using a specific sequence of the S. aureus-specific staphylococcal protein A (spa); SFPO (staphylococcal food poisoning outbreaks

 1

	Strain	Genotype	SET1-PCR	SET2-PCR	Vidas SET2	SET-RPLA	spa-type	Comment
	a	C	c	g, i	+	C+	t529
								Involved	in
	b	B	a, d, r	j	+	A+ D+	t2953	SFPO
	c	B	a	-	+	A+	t5271
	d	B	d, r	j	+	D+	t2953
	e	O	d	g, i	+	D+	t7013	Rare genotype

Table 2 . Thermostat settings used for the different scalding procedures, using a heating rate of 0.5 °C/min. The final temperature was 1° C higher than the target to compensate for the temperature differences between the water bath and the milk temperature

 2

	Scalding	Heating to scalding temperature (heating rate 0.5° C/min)	Hold time scalding
	temperature (°C)		Time		Time
		Temperature (°C)		Temperature (°C)
			(min)		(min)
	46	47	32	46.5	10
	48	49	35	48.5	10
	50	51	39	50.5	10
	52	53	40	52.5	10
	54	55	45	54.5	10
	56	57	48	56.5	10

Table 3 . Results of the SE production by different staphylococcal strains (qualitative measurements of SEA- SEE, Vidas SET2) in cheese 20 h after production. The results are the proportion of positive results out of five cheeses produced at five different scalding temperatures (46-56 °C) and five different strains (a-e, for details, see Table 2

 3

Table S5 :

 S5 Raw Data mRNA [copies/cell] during cell growing T0-T2

	Sampling
	point	seg	seg std	sei	sei std	sem	sem std	sen	sen std	seo	seo std
	T0	2.70E-01	1.35E-01	5.59E-01	2.79E-01	5.58E-01	2.79E-01	8.55E-01	4.28E-01	0.00E+00	0.00E+00
	T1	4.05E+00	3.33E-02	9.69E+00	2.06E+00	1.75E+01	4.82E+00	9.31E+00	2.12E+00	3.96E-01	5.89E-02
	T2	1.05E+00	1.79E-01	2.67E-01	7.61E-02	8.75E-01	1.65E-01	7.66E-01	2.12E-01	0.00E+00	0.00E+00
	T0	6.74E+00	3.83E+00	7.43E+00	4.15E+00	1.32E+01	7.51E+00	6.76E+00	4.13E+00	6.48E-01	3.24E-01
	T1	1.48E+01	8.96E+00	2.11E+01	1.10E+01	4.55E+01	2.59E+01	1.54E+01	8.47E+00	4.27E+01	2.90E+01
	T2	1.93E+00	7.20E-01	1.02E+00	3.27E-01	2.51E+00	8.61E-01	6.53E-01	2.11E-01	8.39E+00	4.16E+00
	T0	0.00E+00	0.00E+00	3.11E+00	0.00E+00	1.45E+00	0.00E+00	8.21E-01	0.00E+00	0.00E+00	0.00E+00
	T1	0.00E+00	0.00E+00	5.30E+01	2.18E+01	6.86E+01	2.10E+01	7.31E+00	3.20E+00	1.90E+00	9.84E-01
	T2	0.00E+00	0.00E+00	4.81E-01	2.40E-01	4.88E-01	1.01E-01	1.53E-01	3.57E-02	2.36E-02	0.00E+00
	T0	4.49E+00	7.56E-01	1.21E+01	1.32E+00	1.83E+01	2.02E+00	9.20E+00	1.55E+00	3.70E-01	5.04E-02
	T1	9.20E+01	2.73E+01	1.32E+02	4.01E+01	2.34E+02	5.84E+01	7.05E+01	1.56E+01	4.99E+01	3.33E+01
	T2	1.72E+00	7.46E-01	1.46E+00	4.57E-01	3.00E+00	8.99E-01	1.17E+00	3.56E-01	1.04E+01	7.32E+00
	T0	6.94E-01	0.00E+00	1.71E+00	0.00E+00	1.24E+00	0.00E+00	9.81E+00	0.00E+00	0.00E+00	0.00E+00
	T1	1.49E+01	5.83E+00	4.51E+01	8.52E+00	4.53E+01	9.35E+00	3.73E+01	9.26E+00	8.03E-01	1.37E-01
	T2	2.20E+00	7.81E-01	3.11E+00	9.59E-01	3.54E+00	1.24E+00	9.96E-01	1.47E-01	8.55E+00	6.01E+00
	T0	0.00E+00	0.00E+00	5.85E+00	3.50E+00	8.17E+00	4.29E+00	6.42E+00	3.62E+00	2.82E+00	1.80E+00
	T1	0.00E+00	0.00E+00	1.44E+01	5.14E+00	2.76E+01	1.22E+01	2.26E+01	1.12E+01	8.07E+01	5.09E+01
	T2	0.00E+00	0.00E+00	1.80E+00	3.37E-01	2.40E+00	4.44E-01	3.04E+00	3.79E-01	8.88E-01	2.23E-01
	T0	3.49E+00	3.80E-01	7.16E+00	4.08E-01	7.48E+00	9.57E-01	3.61E+00	1.08E+00	0.00E+00	0.00E+00
	T1	2.05E+01	4.79E-01	5.90E+01	1.85E-01	8.04E+01	1.42E+00	4.70E+01	6.72E+00	1.57E+00	8.50E-02
	T2	3.84E+00	1.18E+00	3.98E+00	8.65E-01	5.53E+00	1.09E+00	2.78E+00	7.02E-01	9.66E-02	2.81E-02
	T0	1.87E+00	5.59E-01	5.52E+00	1.12E+00	5.02E+00	1.00E+00	3.79E+00	4.84E-01	1.03E+00	1.88E-01
	T1	6.27E+00	3.12E-01	4.91E+01	1.07E+01	4.84E+01	5.47E+00	7.61E+01	7.82E+00	1.72E+01	2.16E-01
	T2	1.95E+00	4.27E-01	2.24E+00	2.83E-01	4.28E+00	3.18E-01	2.18E+00	1.75E-01	6.83E-01	4.17E-02
				XXX

Table S6 :

 S6 Raw Data enterotoxin G and I [ng/ml] during cell growing T0-T2

		Sampling
	Strain	point	SEG	SEGstd	SEI	SEIstd
		T1	1.62	0.17	18.41	5.74
	Mu50	T2	2.66	0.20	61.73	4.13
		T3	3.54	0.28	54.36	4.50
		T1	1.14	0.06	34.53	2.88
	KS24	T2	2.27	0.09	66.86	2.60
		T3	4.24	0.32	70.59	1.25
		T1	0.00	0.00	-	-
	AG	T2	0.00	0.00	13.42	0.21
		T3	0.00	0.00	14.46	0.05
		T1	3.41	0.24	64.58	5.15
	13ceb	T2	7.00	0.23	106.58	2.39
		T3	6.20	0.10	109.41	4.18
		T1	0.51	0.07	2.48	0.24
	26.3	T2	2.30	0.04	6.41	0.32
		T3	4.11	0.08	6.05	0.25
		T1	0.00	0.00	0.00	0.00
	26.1	T2	0.00	0.00	0.00	0.00
		T3	0.00	0.00	0.00	0.00
		T1	0.61	0.05	28.94	7.66
	15SB	T2	3.70	0.04	67.14	0.73
		T3	4.39	0.08	55.17	0.60
		T1	0.23	0.02	3.87	2.06
	13	T2	1.29	0.03	2.67	0.01
		T3	1.99	0.04	1.59	0.14
		T1	0.00	0.00	0.00	0.00
	26.1	T2	0.00	0.00	0.00	0.00
		T3	0.00	0.00	0.00	0.00
		T1	0.51	0.07	2.48	0.24
	26.3	T2	2.30	0.04	6.41	0.32
		T3	4.11	0.08	6.05	0.25
		T1	3.41	0.24	64.58	5.15
	13ceb	T2	7.00	0.23	106.58	2.39
		T3	6.20	0.10	109.41	4.18
		T1	0.61	0.05	28.94	7.66
	15SB	T2	3.70	0.04	67.14	0.73
		T3	4.39	0.08	55.17	0.60
		T1	1.14	0.06	34.53	2.88
	KS24	T2	2.27	0.09	66.86	2.60
		T3	4.24	0.32	70.59	1.25
		T1	1.62	0.17	18.41	5.74
	Mu50	T2	2.66	0.20	61.73	4.13
		T3	3.54	0.28	54.36	4.50
		T1	0.00	0.00	-	-
	AG	T2	0.00	0.00	13.42	0.21
		T3	0.00	0.00	14.46	0.05
				XXXI

Table S7 :

 S7 Raw Data mRNA [copies/cell] at T1 with different NaCl concentrations.

	Strain	NaCl [g/L]	seg	seg std	sei	sei std sem	sem std	sen	sen std	seo	seo std
	KS24	5	33.14	19.46	58.39	35.02	107.95 50.33	69.68	49.00	2.45	1.01
	KS24	12.5	17.09	6.23	22.35	3.85	68.25	24.94	47.98	17.33	1.29	0.41
	KS24	20	18.14	14.52	28.92	16.21	78.26	59.34	44.50	28.24	1.68	1.42
	KS24	100	4.66	0.99	3.82	1.18	11.19	6.90	4.18	2.13	0.36	0.22
	Mu50	5	10.44	4.29	6.39	3.80	15.02	4.36	11.82	7.02	0.46	0.28
	Mu50	12.5	7.32	8.04	3.13	2.06	22.14	23.74	7.17	6.67	0.34	0.24
	Mu50	20	7.40	9.32	3.04	3.37	19.26	24.42	8.44	9.66	0.36	0.28
	Mu50	100	1.48	0.70	0.27	0.09	1.31	0.22	0.54	0.27	0.07	0.06
	15SB	5	26.42	13.21	41.82	14.54	110.49 35.52	31.62	5.78	1.54	0.33
	15SB	12.5	58.51	13.16	71.13	30.84	162.24 47.22	64.25	34.39	2.14	1.32
	15SB	20	17.42	7.12	17.49	8.25	70.55	37.56	20.90	7.78	0.86	0.39
	15SB	100	13.79	13.28	5.55	5.25	24.04	22.32	4.44	3.77	0.43	0.38

Table S8 :

 S8 Raw Data enterotoxin G and I [ng/ml] at T3 with different NaCl concentrations.

	Strain	NaCl [g/L]	SEG	SEGstd	SEI	SEIstd
	KS24	5	70.59	2.50	4.24	0.63
	KS24	12.5	30.60	1.27	2.71	0.46
	KS24	20	35.30	0.23	3.02	0.31
	KS24	100	10.55	2.46	0.50	0.13
	Mu50	5	54.36	9.00	3.54	0.55
	Mu50	12.5	28.54	4.47	1.83	0.32
	Mu50	20	23.72	3.71	1.50	0.24
	Mu50	100	6.08	0.79	0.09	0.01
	15SB	5	55.17	1.20	4.39	0.15
	15SB	12.5	37.64	1.49	2.78	0.09
	15SB	20	34.78	5.87	3.01	0.33
	15SB	100	11.73	4.25	0.58	0.20
				XXXII

Table S9 :

 S9 Table S7: Raw Data mRNA [copies/cell] at T1 with 37 and 45 °C.

	Strain	Temperature [°C]	seg	seg std	sei	sei std	sem	sem std	sen	sen std	seo	seo std
	KS24	37°C	33.14 19.46 58.39 35.02 107.95 50.33 69.68 49.00 2.45	1.01
	KS24	45°C	5.82	2.50	4.98	1.93	15.00	5.62	7.75	3.34	0.71	0.25
	Mu50	37°C	10.44 4.29	6.39	3.80	15.02	4.36	11.82 7.02	0.46	0.28
	Mu50	45°C	8.55	5.65	3.69	3.70	25.62	24.18 7.34	7.87	0.68	0.55
	15SB	37°C	26.42 13.21 41.82 14.54 110.49 35.52 31.62 5.78	1.54	0.33
	15SB	45°C	23.29 23.13 7.42	7.36	38.75	38.18 9.06	8.92	0.82	0.81

Table S10 :

 S10 Raw Data enterotoxin G and I [ng/ml] at T3 with with 37 and 45 °C.

	Strain	Temperature [°C]	SEG	SEGstd	SEI	SEIstd
	KS24	37°C	70.59	2.50	4.24	0.63
	KS24	45°C	4.03	0.87	0.45	0.16
	Mu50	37°C	54.36	9.00	3.54	0.55
	Mu50	45°C	15.13	3.30	0.53	0.12
	15SB	37°C	55.17	1.20	4.39	0.15
	15SB	45°C	6.45	1.49	0.34	0.07

Titre : Evaluation and prediction of staphylococcal enterotoxins Mots-clés :

  Staphylococcus aureus, staphylococcal enterotoxins, enterotoxin gene cluster (egc), SEG, SEI, prediction, stress parameters Outre les infections qu'il peut causer chez l'homme et les animaux, le Staphylococcus aureus peut produire différentes entérotoxines dans les aliments. Ces entérotoxines peuvent provoquer des vomissements et des diarrhées, qui touchent souvent de nombreuses personnes et provoquent ce que l'on appelle des toxi-infections collectives (TIAC) à staphylocoques. À ce jour, de nombreux cas de TIAC sont peu patents, ceci pour de nombreuses raisons. L'une d'elles pourrait être qu'actuellement, seulement cinq entérotoxines, dites classiques, sur les 26 entérotoxines staphylococciques connues, 'est pourquoi de nouveaux outils, tels que le séquençage du génome complet (WGS) et de nouvelles méthodes de mesure des entérotoxines, sont nécessaires afin de pouvoir isoler et caractériser les souches de S. aureus et leurs entérotoxines et, par conséquent, de pouvoir prédire la production d'entérotoxines sur la base des données du génome. C'était le principal objectif de cette étude.Afin d'y parvenir, les entérotoxines egc ont été étudiées au niveau du génome (seg, sei, sem, sen, seo et seu), au niveau de l'expression de l'ARNm (seg, sei, sem, sen, seo) et au niveau des protéines (SEG et SEI). Avec les résultats, nous démontrons que la production in vitro de SEG et de SEI peut être prédite sur la base du type de vSaβ et du clonal complex (CC) d'une souche. De plus, le type de vSaβ/CC permet de prédire la source d'une souche TIAC positive à l'egc (d'origine animale ou humaine).La production d'entérotoxines (SEG et SEI) dans des conditions de stress a également été testée, montrant que les stress liés au NaCl et à la température, imitant les conditions rencontrées dans la production alimentaire, ne semblent pas affecter la production d'entérotoxines egc, SEG et SEI.La présente étude peut contribuer à mieux comprendre l'implication des souches de S. aureus produisant les énterotoxine egc dans les toxi-infections alimentaires collectives à staphylocoques. En outre, les résultats donnent également un premier aperçu des conditions et des limites de la production d'entérotoxines egc, ainsi qu'une première indication sur leur capacité à être produites dans la fabrication et le stockage des aliments.

	Résumé :

peuvent être analysées à l'aide de kits disponibles dans le commerce. Une série d'entérotoxines non classiques (seg, sei, sem, sei, seo et seu) est située sur le même cluster (enterotoxin gene cluster, egc) qui fait partie de l'îlot génomique vSaβ de S. aureus. Ces entérotoxines ont été décrites comme pouvant être impliquées dans des toxiinfections alimentaires collectives à staphylocoques. C

Schwendimann, L.; Mérda, D., Berger, T.; Denayer, S.; Feraudet-Tarisse, C.; Kläui, A.; Messio, S.; Mistou, M.Y.; Nia, Y.; Hennekinne, J.A., Graber, H.U. (2021). Staphylococcal enterotoxin gene cluster: prediction of enterotoxin (SEG and SEI) production and of the source of food poisoning based on vSaβ typing. Appl Env Micr. 10.1128/AEM.02662-20.

Provisional data, additional revision of the manuscript and data needed before submission to a peer reviewed journal

Acknowledgments

This work was financially supported by the Agroscope research program. The work is a collaboration project between Anses and Agroscope. The development of the ELISA method for G and I was funded in part by the French joint ministerial program of R&D against CBRNE threats. In addition, a particular thanks goes to L. Fritsch for the support in using R, as well as to N. Vingadassalon and I. Ivanovic for the help in their laboratories.

Acknowledgments

The authors would like to thank Patrick Bischof, Adrian Burgunder, Desirée Dürr, Elisabeth Eugster, Pierre Gerber, Dieter Weik, and Manuel Wittwer for their help in different parts of the project and Simon Briner for the help in writing the manuscript.

Data availability

Sequencing data for all isolates analyzed in this study have been deposited in the NCBI GenBank under BioProject accession numbers PRJNA633807. Accession numbers for individual genomes and assembly statistics are listed in Appendix II Table S2-S3

Abstract

According to the European Food Safety Authority (2019), 77 out of 114 outbreaks caused by staphylococcal enterotoxin (SFPO) are weak evidence outbreaks. However, only five out of 26 enterotoxins can be analyzed using commercially available kits. Especially the presence of the so called "new enterotoxins", cannot be identified. A group of these new enterotoxin genes are seg, sei, sem, sei, seo and seu that are located on the same enterotoxin gene cluster (egc) and seem to be involved in SFPOs.

The aim of the present study is to improve the understanding of the parameters and conditions under which egc enterotoxins are produced. This knowledge will lead to an improved control of their expression during food production and storage. For this purpose, a selection of eight strains from different origin was chosen based on their genetic diversity (genetic structure) and origin (human, animal, environment, food). Their expression (mRNA) of seg, sei, sem, sen and seo was measured using RT-qPCR at three different points during the bacterial growing phase (start, mid-log and end-log). Based on these results three strains were selected to study their enterotoxin expression under stress conditions: NaCl concentrations up to 100 g/l, higher temperature (45 °C). In addition each sample was tested on Staphylococcal enterotoxins G and I using an in-house sandwich ELISA method. The results show that egc enterotoxins are mostly expressed in the mid-log phase of bacterial growth and seem to switch off at the end of the log phase. SEG and SEI are already produced at an early stage of the growing phase.

Interestingly, common NaCl stress, mimicking conditions in food production, seems not to affect the expression and production of egc enterotoxins up to 20 g/L. However, differences between strains were observed.

The study gives insights into the production of egc enterotoxins under stress conditions. This information will enhance the availability of methods controlling egc enterotoxins in food production and storage.

Keywords: Enterotoxin G, Enterotoxin I, Staphylococcus aureus, NaCl, gene expression

Effect of temperature on the expression and production of egc enterotoxins

For the measurement of the effect of temperature on the expression and production of enterotoxin, this was measured for three strains (Mu50, 15SB, KS24) at standard conditions (37 °C) and at 45 °C (Figure 30).

Statistical analysis data

Robust linear regression for verify the regression model and to identify outliers