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National audience; La pollution plastique de tous les compartiments de l’environnement, y compris l’environnement marin, n’est désormais plus à démontrer. Concomitamment à cette pollution, la présence de particules plastiques de tailles micrométriques et nanométriques a également été rapportée. Ces particules peuvent constituer un danger physique ou agir en tant que vecteurs de différents contaminants chimiques ou biologiques. Chez les organismes vivants le devenir de ces particules n’est que partiellement connu et le passage des barrières biologiques reste controversé, bien qu’il soit probable pour les particules les plus petites comme les nanoplastiques. Afin de répondre à cette question de la possible translocation de nanoplastiques, des manipulations d’exposition ex vivo à des nanobilles de polystyrène modèle d’une taille de 50 nm (NP-PS) ont été réalisées sur des intestins de bar commun (Dicentrarchus labrax) en utilisant des chambres de Ussing.Grâce à la combinaison des résultats obtenus par plusieurs techniques de détection : fluorométrie, microscopie électronique à balayage (MEB) et pyrolyse couplée à la chromatographie en phase gazeuse et à un spectromètre de masse haute-résolution (Py-GC-HRMS), il a été possible d’enregistrer une augmentation de la fluorescence du côté séreux quelques dizaines de minutes après l’exposition des échantillons à la solution de NP-PS. Cette mesure a pu être confirmée par celles réalisées en MEB et Py-GC-HRMS. La translocation de nanobilles de PS de 50nm et sans agrégation dans le milieu expérimental, à travers la paroi de l’intestin de cette espèce de poisson a donc été attestée.

International audience; This opinion paper offers a scientific view on the current debate of the place of biodegradable plastics as part of the solution to deal with the growing plastic pollution in the world's soil, aquatic, and marine compartments. Based on the current scientific literature, we focus on the current limits to prove plastic biodegradability and to assess the toxicity of commercially used biobased and biodegradable plastics in natural environments. We also discuss the relevance of biodegradable plastics for selected applications with respect to their use and end of life. In particular, we underlined that there is no universal biodegradability of plastics in any ecosystem, that considering the environment as a waste treatment system is not acceptable, and that the use of compostable plastics requires adaptation of existing organic waste collection and treatment channels.

International audience; Listeria monocytogenes can grow under stressful conditions and contaminate various food categories. Progresss in DNA sequencing-based identification methods, such as multi-locus sequence typing (MLST) now allow for more accurate characterization of pathogens. L. monocytogenes MLST genetic diversity is reflected by the different prevalence of the "clonal complexes" (CCs) in foods or infections. Better understanding of the growth potentials of L. monocytogenes is essential for quantitative risk assessment and efficient detection across CCs genetic diversity. Using optical density measurements taken with an automated spectrophotometer, we compared the maximal growth rate and lag phase of 39 strains from 13 different CCs and various food origins, in 3 broths mimicking stresful food conditions (8 °C, aw 0.95 and pH5) and in ISO Standard enrichment broths (Half Fraser and Fraser). This is important as growth could influence risk through pathogen multiplication in food. Besides, enrichment problems could lead to a lack of detection of some CCs. Despite small differences highlighting natural intraspecific variability, our results show that growth performances of L. monocytogenes strains under the conditions tested in selective and non-selective broth do not appear to be strongly correlated to CCs and cannot explain higher CC "virulence" or prevalence.

Raw reads of the 34 genomes of S. Bovismorbificans and 12 genomes of S. Typhimurium from human and food origin in this study were submitted to the European Nucleotide Archive under project numbers PRJEB41645 and PRJEB49565.; International audience; The French National Reference Centre for Escherichia coli, Shigella and Salmonella (FNRC-ESS) detected two human clusters of 33 cases (median age: 10 years; 17 females) infected by Salmonella enterica serotype Bovismorbificans, ST142, HC5_243255 (EnteroBase HierCC‑cgMLST scheme) in September-November 2020 and of 11 cases (median age: 11 years; seven males) infected by S. enterica serotype 4,12:i:-, ST34, HC5_198125 in October-December 2020. Epidemiological investigations conducted by Santé publique France linked these outbreaks to the consumption of dried pork sausages from the same manufacturer. S. Bovismorbificans and S. 4,12:i:- were isolated by the National Reference Laboratory from different food samples, but both strains were identified in a single food sample only by qPCR. Three recalls and withdrawals of dried pork products were issued by the French general directorate of food of the French ministry for agriculture and food in November 2020, affecting eight supermarket chains. A notification on the European Rapid Alert System for Food and Feed and a European urgent enquiry on the Epidemic Intelligence Information System for Food and Waterborne Diseases and Zoonoses (EPIS-FWD) were launched. No cases were reported outside France. Outbreaks caused by multiple serotypes of Salmonella may go undetected by protocols in standard procedures in microbiology laboratories.

Publication on Microorganisms. 11 January 2023. https://www.mdpi.com/2076-2607/11/1/177 Abstract: Mammalian tuberculosis (TB) is a zoonotic disease mainly due toMycobacterium bovis(M. bovis). A current challenge for its eradication is understanding its transmission within multi-host systems. Improvements in long-read sequencing technologies have made it possible to obtain complete bacterial genomes that provide a comprehensive view of species-specific genomic features. In the context of TB, new genomic references based on complete genomes genetically close to field strains are also essential to perform precise field molecular epidemiological studies. A total of 10M. bovisstrains representing each genetic lineage identified in France and in other countries were selected for performing complete assembly of their genomes. Pangenome analysis revealed a “closed” pangenome composed of 3900 core genes and only 96 accessory genes. Whole genomes-based alignment using progressive Mauve showed remarkable conservation of the genomic synteny except that the genomes have a variable number of copies of IS6110. Characteristic genomic traits of each lineage were identified through the discovery of specific indels. Altogether, these results provide new genetic features that improve the description ofM. bovislineages. The availability of new complete representative genomes ofM. boviswill be useful to epidemiological studies and better understand the transmission of this clonal-evolving pathogen.

International audience; Tick-borne encephalitis (TBE) is a viral disease endemic in Eurasia. The virus is mainly transmitted to humans via ticks and occasionally via the consumption of unpasteurized milk products. The European Centre for Disease Prevention and Control reported an increase in TBE incidence over the past years in Europe as well as the emergence of the disease in new areas. To better understand this phenomenon, we investigated the drivers of TBE emergence and increase in incidence in humans through an expert knowledge elicitation. We listed 59 possible drivers grouped in eight domains and elicited forty European experts to: (i) allocate a score per driver, (ii) weight this score within each domain, and (iii) weight the different domains and attribute an uncertainty level per domain. An overall weighted score per driver was calculated, and drivers with comparable scores were grouped into three terminal nodes using a regression tree analysis. The drivers with the highest scores were: (i) changes in human behavior/activities; (ii) changes in eating habits or consumer demand; (iii) changes in the landscape; (iv) influence of humidity on the survival and transmission of the pathogen; (v) difficulty to control reservoir(s) and/or vector(s); (vi) influence of temperature on virus survival and transmission; (vii) number of wildlife compartments/groups acting as reservoirs or amplifying hosts; (viii) increase of autochthonous wild mammals; and (ix) number of tick species vectors and their distribution. Our results support researchers in prioritizing studies targeting the most relevant drivers of emergence and increasing TBE incidence.

International audience

This paper was prepared thanks to the collective expertise carried out by the ANSES emergency collective expert appraisal group (GECU) “Monkeypox – Food” (ANSES opinion no 2022-SA-0110), chaired by Nadia Haddad, and whose members are cited in the authors.; International audience; Monkeypox (MPX) is a zoonotic infectious disease caused by Monkeypox virus (MPXV), an enveloped DNA virus belonging to the Poxviridae family and the Orthopoxvirus genus. Since early May 2022, a growing number of human cases of Monkeypox have been reported in non-endemic countries, with no history of contact with animals imported from endemic and enzootic areas, or travel to an area where the virus usually circulated before May 2022. This qualitative risk assessment aimed to investigate the probability that MPXV transmission occurs through food during its handling and consumption. The risk assessment used “top-down” (based on epidemiological data) and “bottom-up” (following the agent through the food chain to assess the risk of foodborne transmission to human) approaches, which were combined. The “top-down” approach first concluded that bushmeat was the only food suspected as a source of contamination in recorded cases of MPXV, by contact or ingestion. The “bottom-up” approach then evaluated the chain of events required for a human to become ill after handling or consuming food. This approach involves several conditions: (i) the food must be contaminated with MPXV (naturally, by an infected handler or after contact with a contaminated surface); (ii) the food must contain viable virus when it reaches the handler or consumer; (iii) the person must be exposed to the virus and; (iv) the person must be infected after exposure. Throughout the risk assessment, some data gaps were identified and highlighted. The conclusions of the top-down and bottom-up approaches are consistent and suggest that the risk of transmission of MPXV through food is hypothetical and that such an occurrence was never reported. In case of contamination, cooking (e.g., 12 min at 70°C) could be considered effective in inactivating Poxviridae in foods. Recommendations for risk management are proposed. To our knowledge, this is the first risk assessment performed on foodborne transmission of MPXV.

International audience; Les additifs plastiques, ajoutés aux polymères au cours de leur formulation, participent aux dangers que posent l’utilisation et le rejet de plastique dans l’environnement. Cet impact dit « chimique » a été démontré à de multiples reprises. En effet, une fois dans l’environnement, les additifs plastiques peuvent être libérés de la matrice polymérique et diffuser dans l’eau environnante ou au sein des organismes après ingestion de particules de plastiques. L’utilisation de certains additifs est controversée car ils se sont révélés être toxiques pour l’environnement, les organismes marins et l’Homme. Afin d’avoir une meilleure compréhension de l’impact chimique de ces additifs, il est important de les identifier, et idéalement de les quantifier. Après avoir sélectionné une soixantaine de ces additifs (plastifiants, retardateurs de flammes, antioxydants, …), une méthode unique pour l’analyse de l'ensemble ces molécules a été développée en pyrolyse couplée à la chromatographie gazeuse et la spectrométrie de masse haute résolution (Py-GC-HRMS). La méthode développée à ensuite permis d’identifier la présence de divers additifs plastiques dans des échantillons de barquettes plastiques alimentaires en polypropylène (PP) et en acide polylactique (PLA). Au vu des additifs mis en évidence dans ces échantillons, et du fait de la forte présence de barquettes alimentaires dans l’environnement, une étude complémentaire s’est portée sur l’impact potentiel de ces additifs sur les stades de reproduction et les jeunes stades de vies de l’huitre creuse (Crassostrea gigas) (une espèce sentinelle, ingénieure et mondialement commercialisée), après exposition à des lixiviats de ces échantillons.

Listeria monocytogenes (Lm) is a ubiquitous bacterium that causes the serious foodborne illness listeriosis. Although soil is a primary reservoir and a central habitat for Lm, little information is available on the genetic features underlying the fitness of Lm strains in this complex habitat. The aim of this study was to identify (i) correlations between the strains fitness in soil, their origin and their phylogenetic position (ii) identify genetic markers allowing Lm to survive in the soil. To this end, we assembled a balanced panel of 216 Lm strains isolated from three major ecological compartments (outdoor environment, animal hosts, and food) and from 33 clonal complexes occurring worldwide. The ability of the 216 strains to survive in soil was tested phenotypically. Hierarchical clustering identified three phenotypic groups according to the survival rate (SR): phenotype 1 “poor survivors” (SR < 2%), phenotype 2 “moderate survivors” (2% < SR < 5%) and phenotype 3 “good survivors” (SR > 5%). Survival in soil depended neither on strains’ origin nor on their phylogenetic position. Genome-wide-association studies demonstrated that a greater number of genes specifically associated with a good survival in soil was found in lineage II strains (57 genes) than in lineage I strains (28 genes). Soil fitness was mainly associated with variations in genes (i) coding membrane proteins, transcription regulators, and stress resistance genes in both lineages (ii) coding proteins related to motility and (iii) of the category “phage-related genes.” The cumulative effect of these small genomic variations resulted in significant increase of soil fitness.

Citation suggérée : Anses. (2022). Recommandations relatives à la réduction du risque de transmission du virus Monkeypox (MPXV) lié à la manipulation et la consommation des denrées alimentaires. (Saisine 2022-SA-0110). Maisons-Alfort : Anses, 33 p.; Le Monkeypox (MPX ou « variole du singe ») est une maladie infectieuse due à un virus (Monkeypox virus - MPXV) de la famille des Poxviridae et du genre Orthopoxvirus, virus enveloppé à ADN. En France, les infections par ce virus font l’objet d’une surveillance pérenne par le dispositif de la déclaration obligatoire.Depuis le début du mois de mai 2022, de nombreux cas autochtones d’infection à virus Monkeypox (MPXV) ont été signalés dans plusieurs pays non endémiques. Le premier cas d’infection par le virus Monkeypox a été confirmé le 19 mai 2022 en Île-de-France.Ainsi, au 21 juin 2022, 2746 cas humains ont été confirmés dans l’Union européenne/Espace économique européen (UE/EEE)3. A la date du 22 juin, 3308 cas confirmés dans le monde sont signalés dans 42 pays, dont 426 cas hors UE/EEE4. En France, au 23 juin 2022, 330 cas de Monkeypox ont été confirmés5. 52 des 287 cas investigués par Santé publique France sont des cas secondaires. À ce jour, en Europe, ces cas sont survenus sans historique de contact avec un animal importé de zone endémique et enzootique, chez des personnes ne rapportant pas de voyage dans une zone de circulation habituelle du virus, et dans le contexte d’une épidémie à transmission interhumaine exclusive à ce jour.L’Anses a été saisie une première fois le 3 juin 2022 concernant des recommandations relatives à la réduction du risque de diffusion du virus Monkeypox aux animaux en France (saisine n°2022-SA-0102).Dans cette seconde saisine, il est demandé à l’Anses : « d’évaluer le risque de transmission du MPXV par les denrées alimentaires au cours de leur manipulation, et de leur consommation et d’émettre des recommandations relatives à la réduction de ce dernier ».

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

Citation suggérée : Anses. (2022). Avis relatif à l'évaluation des risques liés à la présence de 2-chloroéthanol (2-CE) dans du gluten incorporé dans des denrées alimentaires (saisine 2022-SA-0059). Maisons-Alfort : Anses, 98 p.; Une alerte sanitaire concernant la détection de 2-chloroéthanol (2-CE) dans des mix à base de gluten vital1 a été rapportée le 23 mars 2022 par la DGCCRF. La présence de 2-CE a été détectée par les préparateurs des mix de gluten qui sont destinés à être incorporés dans des farines utilisées pour la fabrication de différents produits finis, dont de panification comme le pain, les viennoiseries, etc. A la suite de cette détection, le fabricant du gluten mis en cause a réalisé des analyses sur son échantillothèque et a confirmé que le gluten était l’ingrédient alimentaire contaminé. Le fabricant a également analysé un ensemble conséquent de plusieurs centaines de lots récents de gluten, amidon et autres coproduits. L’oxyde d’éthylène(ETO) auquel le métabolite 2-CE est communément associé n’a été détecté dans aucun échantillon.Selon la DGCCRF, ces résultats excluent tout lien avec une contamination ou un traitement par l’ETO, traitement interdit en Europe, et seraient ainsi concordants avec l’origine française des blés.Dans ce contexte, il est demandé à l’Anses de mener une expertise en urgence afin de répondre aux questions suivantes :- Quel est le niveau d’exposition alimentaire et le niveau de préoccupation sanitaire associé ?- Pouvez-vous, sur la base des éléments disponibles, conclure sur le niveau de préoccupation sanitaire pour les produits finis au sein desquels le 2-CE n’est pas quantifiable (< 0,02 mg/kg) ?

The authors are grateful to collaborators of the SACADA consortium: Géraldine Boué, Steven Duret, Michel Federighi, Maxence Feher, Lisa Fourniol, Yvonnick Guillois, Pauline Kooh, Sophie Le Poder, Anne-Laure Maillard, Jean-Claude Manuguerra, Mathilde Pivette, Jessica Vanhomwegen, Prunelle Waldman; International audience; SARS-CoV-2 (Severe acute respiratory syndrome coronavirus 2), a virus causing severe acute respiratory disease in humans, emerged in late 2019. This respiratory virus can spread via aerosols, fomites, contaminated hands or surfaces as for other coronaviruses. Studying their persistence under different environmental conditions represents a key step for better understanding the virus transmission. This work aimed to present a reproducible procedure for collecting data of stability and inactivation kinetics from the scientific literature. The aim was to identify data useful for characterizing the persistence of viruses in the food production plants. As a result, a large dataset related to persistence on matrices or in liquid media under different environmental conditions is presented. This procedure, combining bibliographic survey, data digitalization techniques and predictive microbiological modelling, identified 65 research articles providing 455 coronaviruses kinetics. A ranking step as well as a technical validation with a Gage Repeatability & Reproducibility process were performed to check the quality of the kinetics. All data were deposited in public repositories for future uses by other researchers.

The foodborne pathogen, Listeria monocytogenes, (Lm), frequently undergoes selection pressure associated with the extensive use of disinfectants, such as quaternary ammonium compounds, which are widely used in food processing plants. The repeated exposure to sub-inhibitory biocide concentrations can induce increased tolerance to these compounds, but can also trigger the development of antibiotic resistance, and both increase the risk of food contamination and persistence in food production environments. Although the acquisition of genes can explain biocide tolerance, the genetic mechanisms underlying the adaptive cross-resistance to antibiotics remain unclear. We previously showed that repeated exposure to benzalkonium chloride (BC) and didecyldimethyl ammonium chloride (DDAC) led to reduced susceptibility to ciprofloxacin in Lm strains from diverse sources. Here, we compared the genomes of 16 biocide-adapted and 10 parental strains to identify the molecular mechanisms of fluoroquinolone cross-resistance. A core genome SNP analysis identified various mutations in the transcriptional regulator fepR (lmo2088) for 94% of the adapted strains and mutations in other effectors at a lower frequency. FepR is a local repressor of the MATE fluoroquinolone efflux pump FepA. The impact of the mutations on the structure and function of the protein was assessed by performing in silico prediction and protein homology modeling. Our results show that 75% of the missense mutations observed in fepR are located in the HTH domain of the protein, within the DNA interaction site. These mutations are predicted to reduce the activity of the regulator, leading to the overexpression of the efflux pump responsible for the ciprofloxacin-enhanced resistance.

International audience; The marine environment is a potential natural reservoir of antimicrobial resistance genes (ARGs), subject to anthropogenic effluents (wastewater, industrial, and domestic), and known as a final receiving system. The aim of this study was to investigate the abundance and geographical distribution of the three bla TEM , sul1 , and intI1 genes, proposed as indicators of contamination to assess the state of antimicrobial resistance in environmental settings, added to the tetA gene and the microbial population ( tuf gene) in the English Channel and North Sea areas. Bacterial DNA was extracted from 36 seawater samples. The abundance of these genes was determined by quantitative PCR (qPCR) and was analyzed in association with environmental variables and geographical locations to determine potential correlations. The bla TEM and tetA genes were quantified in 0% and 2.8% of samples, respectively. The sul1 and intI1 genes were detected in 42% and 31% of samples, respectively, with an apparent co-occurrence in 19% of the samples confirmed by a correlation analysis. The absolute abundance of these genes was correlated with the microbial population, with results similar to the relative abundance. We showed that the sul1 and intI1 genes were positively correlated with dissolved oxygen and turbidity, while the microbial population was correlated with pH, temperature and salinity in addition to dissolved oxygen and turbidity. The three tetA , sul1 , and intI1 genes were quantified in the same sample with high abundances, and this sample was collected in the West Netherlands coast (WN) area. For the first time, we have shown the impact of anthropogenic inputs (rivers, man-made offshore structures, and maritime activities) and environmental variables on the occurrence of three indicators of environmental contamination by antimicrobial resistance in the North Sea and English Channel seawaters.

National audience; Environmental pollution from plastics and microplastics (MPs) is a threat to the environment and wildlife on multiple aspects regarding exposition of different hazards. One of them is due to the chemical impact of the organic plastic additives (OPAs). Various additives can be added to polymers during the manufacturing process to modify and improve their physical properties. The analysis of OPAs has shown a growing importance since their use has become controversial as some of the additives were found to be toxic to marine organism and humans. In order to have a better understanding of the chemical impact of those additives, it is important to identify and, ideally quantify the additives included in the polymeric matrices. The analysis of OPAs and polymers is an analytical challenge due to the diversity of their chemical composition, the fact that they are most of the time mixed, and the numerous steps needed for the sample preparation with “conventional” approaches such as solvent extraction (SE). Thus, implementing easy and quick screening analysis methods, able to analyse plastic additives, have become a focus of interest. Pyrolysis coupled to a gas chromatography and a high-resolution mass spectrometer (Py-GC-HRMS) is an analytical tool that enable a relatively quick and easy analysis. This technique minimize or even remove the steps for sample preparation, limiting their potential contamination prior the analysis.The goal of this study is to develop a method using Py-GC-HRMS to identify and, if possible, quantify OPAs included in a polymeric matrix. A single method for the analysis of various selected plastic additives has been implemented and benefits the constitution of a database in GC/MS-Orbitrap with a wide variety of plastic additives. This method has shown its ability to characterise several additives is plastic packaging samples. The development of the quantitative method is still on going.

International audience; Characterising pesticide residues from a qualitative and quantitative point of view is key to both risk assessment in the framework of pesticide approval and risk management. In the European Union (EU), these concerns are addressed during the evaluation of active substances at the European level prior to marketing authorisation. In the framework of this review, we will focus on one specific item of the residue section, namely the effect of process (industrial or domestic transformation of the raw commodities) on the nature of the residue in food. A limited number of hydrolysis conditions defined by three parameters (temperature, pH and time) are set to be “representative of the most widely used industrial and domestic food processing technologies”. These hydrolysis conditions, however, do not cover processes at temperatures higher than 120 °C, such as cooking with a conventional oven or in a pan, frying or using a microwave oven.

International audience; Tick-borne encephalitis virus’ (TBEV) geographic range and the human incidence are increasing throughout Europe, putting a number of non-endemic regions and countries at risk of outbreaks. In spring 2020, there was an outbreak of tick-born encephalitis (TBE) in Ain, Eastern France, where the virus had never been detected before. All patients but one had consumed traditional unpasteurised raw goat cheese from a local producer. We conducted an investigation in the suspected farm using an integrative One Health approach. Our methodology included (i) the detection of virus in cheese and milk products, (ii) serological testing of all animals in the suspected farm and surrounding farms, (iii) an analysis of the landscape and localisation of wooded area, (iv) the capture of questing ticks and small mammals for virus detection and estimating enzootic hazard, and (v) virus isolation and genome sequencing. This approach allowed us to confirm the alimentary origin of the TBE outbreak and witness in real-time the seroconversion of recently exposed individuals and excretion of virus in goat milk. In addition, we identified a wooded focus area where and around which there is a risk of TBEV exposure. We provide the first TBEV isolate responsible for the first alimentary-transmitted TBE in France, obtained its full-length genome sequence, and found that it belongs to the European subtype of TBEV. TBEV is now a notifiable human disease in France, which should facilitate surveillance of its incidence and distribution throughout France.

International audience; Bovine Tuberculosis (bTB) is a zoonotic disease due to Mycobacterium bovis (M. bovis). France has a bTB-free status, but the disease has not been eradicated yet and a worryingly steady increase of bTB outbreaks has been observed in some regions (1). This could be explained by the detection of bTB in wildlife that spills it back to livestock in the same territories. The transmission link within these multi-host systems remains difficult to establish given that they share the same M. bovis genotypes (2). Obtaining new reference genomes for each of these genotypes could improve the knowledge of clonal groups and refine molecular field epidemiology studies based on whole genome sequencing.

The ubiquitous and growing presence of microplastics (MPs) in all compartments of the environment raises concerns about their possible harmful effects on human health. Human exposure to MPs occurs largely through ingestion. Polyethylene (PE) is widely employed for reusable bags and food packaging and found to be present in drinking water and food. It is also one of the major polymers detected in human stool. The aim of this study was to characterize the effects of intestinal exposure to PE MPs on gut homeostasis. Mice were orally exposed for 6 weeks to PE microbeads of 2 different sizes, 36 and 116 μm, that correspond to those found in human stool. They were administrated either individually or as a mixture at a dose of 100 μg/g of food. Both PE microbead sizes were detected in mouse stool. Different parameters related to major intestinal functions were compared between control mice, mice exposed to each type of microbead, or co-exposed to the 2 types of microbeads. Intestinal disturbances were observed after individual exposure to each size of PE microbead, and the most marked deleterious effects were found in co-exposed mice. At the histomorphological level, crypt depth was increased throughout the intestinal tissues. Significant variations of gene expression related to epithelial, permeability, and inflammatory biomarkers were quantified. Defective recruitment of some intestinal immune cells was observed from the proximal portion of the small intestine to the colon. Several bacterial taxa at the order level were found to be affected by exposure to the MPs by metagenomic analysis of cecal microbiota. These results show that ingestion of PE microbeads induces significant alterations of crucial intestinal markers in mice and underscores the need to further study the health impact of MP exposure in humans.

Listeria monocytogenes (Lm) is a ubiquitous bacterium that causes listeriosis, a serious foodborne illness. In the nature-to-human transmission route, Lm can prosper in various ecological niches. Soil and decaying organic matter are its primary reservoirs. Certain clonal complexes (CCs) are over-represented in food production and represent a challenge to food safety. To gain new understanding of Lm adaptation mechanisms in food, the genetic background of strains found in animals and environment should be investigated in comparison to that of food strains. Twenty-one partners, including food, environment, veterinary and public health laboratories, constructed a dataset of 1484 genomes originating from Lm strains collected in 19 European countries. This dataset encompasses a large number of CCs occurring worldwide, covers many diverse habitats and is balanced between ecological compartments and geographic regions. The dataset presented here will contribute to improve our understanding of Lm ecology and should aid in the surveillance of Lm. This dataset provides a basis for the discovery of the genetic traits underlying Lm adaptation to different ecological niches.

The massive contamination of the environment by plastics is an increasing global scientific and societal concern. Knowing whether and how these pollutants affect the behaviour of keystone species is essential to identify environmental risks effectively. Here, we focus on the effect of plastic leachates on the behavioural response of the common blue mussel Mytilus edulis, an ecosystem engineer responsible for the creation of biogenic structures that modify the environment and provide numerous ecosystem functions and services. Specifically, we assess the effect of virgin polypropylene beads on mussels' chemotactic (i.e. a directional movement in response to a chemical stimulus) and chemokinetic (i.e. a non-directional change in movement properties such as speed, distance travelled or turning frequency in response to a chemical stimulus) responses to different chemical cues (i.e. conspecifics, injured conspecifics and a predator, the crab Hemigrapsus sanguineus). In the presence of predator cues, individual mussels reduced both their gross distance and speed, changes interpreted here as an avoidance behaviour. When exposed to polypropylene leachates, mussels moved less compared to control conditions, regardless of the cues tested. Additionally, in presence of crab cues with plastic leachates, mussels significantly changed the direction of movement suggesting a leachate-induced loss of their negative chemotaxis response. Taken together, our results indicate that the behavioural response of M. edulis is cue-specific and that its anti-predator behaviour as well as its mobility are impaired when exposed to microplastic leachates, potentially affecting the functioning of the ecosystem that the species supports. info:eu-repo/semantics/publishedVersion

International audience; Plastic pollution in marine ecosystems constitutes an important threat to marine life. For vertebrates, macro/microplastics can obstruct and/or transit into the airways and digestive tract whereas nanoplastics (NPs; < 1000 nm) have been observed in non-digestive tissues such as the liver and brain. Whether NPs cross the intestinal epithelium to gain access to the blood and internal organs remains controversial, however. Here, we show directly NP translocation across the intestinal barrier of a fish, the European seabass, Dicentrarchus labrax, ex vivo. The luminal side of median and distal segments of intestine were exposed to fluorescent polystyrene NPs (PS-NPs) of 50 nm diameter. PS-NPs that translocated to the serosal side were then detected quantitatively by fluorimetry, and qualitatively by scanning electron microscopy (SEM) and pyrolysis coupled to gas chromatography and high-resolution mass spectrometry (Py-GC-HRMS). Fluorescence intensity on the serosal side increased 15–90 min after PS-NP addition into the luminal side, suggesting that PS-NPs crossed the intestinal barrier; this was confirmed by both SEM and Py-GC-HRMS. This study thus evidenced conclusively that NPs beads translocate across the intestinal epithelium in this marine vertebrate.

International audience; Plastic food packaging represents 40 % of the plastic production worldwide and belongs to the 10 most commonly found items in aquatic environments. They are characterized by high additives contents with >4000 formulations available on the market. Thus they can release their constitutive chemicals (i.e. additives) into the surrounding environment, contributing to chemical pollution in aquatic systems and to contamination of marine organism up to the point of questioning the health of the consumer. In this context, the chemical and toxicological profiles of two types of polypropylene (PP) and polylactic acid (PLA) food packaging were investigated, using in vitro bioassays and target gas chromatography mass spectrometry analyses. Plastic additives quantification was performed both on the raw materials, and on the material leachates after 5 days of lixiviation in filtered natural seawater. The results showed that all samples (raw materials and leachates) contained additive compounds (e.g. phthalates plasticizers, phosphorous flame retardants, antioxidants and UV-stabilizers). Differences in the number and concentration of additives between polymers and suppliers were also pointed out, indicating that the chemical signature cannot be generalized to a polymer and is rather product dependent. Nevertheless, no significant toxic effects was observed upon exposure to the leachates in two short-term bioassays targeting baseline toxicity (Microtox® test) and Pacific oyster Crassostrea gigas fertilization success and embryo-larval development. Overall, this study demonstrates that both petrochemical and bio-based food containers contain harmful additives and that it is not possible to predict material toxicity solely based on chemical analysis. Additionally, it highlights the complexity to assess and comprehend the additive content of plastic packaging due to the variability of their composition, suggesting that more transparency in polymer formulations is required to properly address the risk associated with such materials during their use and end of life.

International audience; Chemical contaminant concentrations in wild organisms are used to assess environmental status under the Eu-ropean Marine Strategy Framework Directive. However, this approach is challenged by the complex intra- andinter-species variability, and the different regional features. In this study, concentrations in trace elements (As,Cd, Hg and Pb), polychlorinated biphenyls (PCBs), polychlorodibenzo-para-dioxines (PCDDs) and poly-chlorodibenzofuranes (PCDFs) were monitored in 8 fish species sampled on the continental shelf of three Frenchregions: the Eastern English Channel (EEC) and Bay of Biscay (BoB) in the Northeast Atlantic Ocean, and the Gulfof Lions (GoL) in Western Mediterranean Sea. Our objectives were to identify species or regions more likely to becontaminated and to assess how to take this variability into account in environmental assessment. While con-centrations were higher in benthic and demersal piscivores, PCB and PCDD/F concentrations (lipid-weight) weresimilar in most teleost species. For Cd, Hg and Pb, the trophic group accumulating the highest concentrationsdepended on the contaminant and region. Concentrations in Hg, PCBs and PCDD/Fs were higher in the EEC and/or GoL than in BoB. Cadmium and Pb concentrations were highest in the BoB. Lipid content accounted for 35%–84% of organic contaminant variability. Lipid normalisation was employed to enhance robustness in the iden-tification of spatial patterns. Contaminant patterns in chondrichthyans clearly differed from that in teleosts. Inaddition, trophic levels accounted for ≤1% and ≤33% of the contaminant variability in teleost fishes in the EECand BoB, respectively. Therefore, developing taxa-specific thresholds might be a more practical way forward forenvironmental assessment than normalisation to trophic levels

Staphylococcal food poisoning outbreaks (SFPO) are caused by the ingestion of food contaminated with staphylococcal enterotoxins (SE), produced by certain strains of coagulase-positive staphylococci. To date, 35 SE are described in the literature (SEA to SEl33) but only five types, SEA to SEE, are routinely detectable via the EN ISO 19020 standard or ELISA (Enzyme-Linked Immunosorbent Assay) techniques because specific antibodies are not available for all SE. During an SFPO, it regularly happens that no SEA-SEE type SE is found even though a typical symptomatology of staphylococcal poisoning has been identified. In addition, epidemiological data highlight the frequent presence of other seg, seh and sei type genes in S. aureus strains derived from SFPO. It is therefore necessary to develop analytical and confirmatory tools targeting a wide range of SE. Mass spectrometry (MS) provides a high specificity in complex media and ability for multiplex analyses. Moreover, it does not necessarily requires the production of specific antibodies for each SE (except for particular formats, such as immunocapture). Thus, MS appears to be a relevant alternative, particularly for the detection of known or recently described SE for which no specific antibodies exist.During this doctoral thesis, a very sensitive MS method was developed first for the quantification and characterization of the first eight SE (SEA-SEE and SEG-SEI) in food matrix. This method uses an extraction step of SE by immunocapture (commercial or CEA antibodies) followed by a trypsin digestion (bottom-up). The specificity of our method allowed the detection of all eight SE in food matrices. This method was optimized to reach a limit of detection between 0.1 and 0.5 ng/g. These concentrations correspond to the concentrations found during SFPO episodes. This MS method was successfully applied to a variety of SFPO matrices, giving results consistent with available ELISA and PCR (Polymerase Chain Reaction) tests. In the meantime, in order to obtain more information on variants and their sequence, a top-down analysis (intact protein) was developed. The top-down analysis allows a global analysis of the sequence of the targeted SE as opposed to the bottom-up mode which only detects certain peptides. An optimization of MS parameters and data processing was performed and an internal standard was selected. This allowed us to obtain an absolute quantification method for SEA with a sensitivity close to the bottom-up technique. To illustrate the interest of this new method, it was applied to 13 SEA variant-producing strains, in parallel with genomic analyses. Finally, we decided to extend the quantification beyond the first eight SE. Thus, we optimized a sample preparation method that does not use antibodies. The method was applied to SE produced by S. aureus strains in culture medium. We were able to identify 24 SE types and more than 162 variants for the first time. Peptides specific of each type and common to all variants were identified. Subsequently, a "label free" quantification method was proposed, based on the "Hi3" protocol, which consists in using the three most intense peptides of each SE for the quantification. By using this method, we were able to identify the ES produced in high quantity and to establish their kinetics of production by S. aureus in culture medium, giving new indications on the priority SE during SFPO., Les toxi-infections alimentaires collectives (TIAC) à staphylocoques sont causées par l'ingestion d’aliments contaminés par des entérotoxines staphylococciques (ES), produites par certaines souches de staphylocoques à coagulase positive. À ce jour, 35 ES sont décrites dans la littérature (SEA à SEl33) mais seules cinq types, SEA à SEE, sont détectables en routine via la norme EN ISO 19020 ou via des techniques ELISA (Enzyme-Linked Immunosorbent Assay) car des anticorps spécifiques ne sont pas disponibles pour l’ensemble des ES. Lors d’épisodes de TIAC, il arrive régulièrement qu’aucune ES de type SEA-SEE ne soit retrouvée alors qu’une symptomatologie typique d’intoxication à staphylocoques a été identifiée. De plus, les données épidémiologiques soulignent la présence fréquente d’autres gènes types seg, seh et sei dans des souches de S. aureus issus de TIAC. Il est donc impératif de disposer d’outils d’analyse et de confirmation capables de viser une large gamme d’ES. La spectrométrie de masse (SM) apporte une grande spécificité d’analyse au sein de milieux complexes et une capacité élevée de multiplexage des analyses. D’autre part, elle ne nécessite pas obligatoirement la production d’anticorps spécifiques de chaque ES (sauf formats particuliers, type immunocapture). Ainsi, la SM apparaît comme une alternative pertinente, particulièrement pour la détection des ES connues ou récemment décrites pour lesquelles il n’existe pas d’anticorps spécifiques. Lors de cette thèse, une méthode par SM a tout d’abord été développée pour la quantification et la caractérisation des huit premières ES (SEA-SEE et SEG-SEI) en matrice alimentaire. Cette méthode utilise l’approche analytique la plus sensible d’après la littérature, c.a.d. une extraction des ES par immunocapture (anticorps commerciaux ou produits au CEA) suivie d’une digestion trypsique (bottom-up). La spécificité de notre méthode a permis de détecter les huit ES dans des matrices alimentaires, et nous avons pu atteindre une limite de détection comprise entre 0,1 et 0,5 ng/g. Ces concentrations correspondent aux concentrations retrouvées lors des investigations d’épisodes de TIACs. Cette méthode LCMS a été appliquée avec succès à des matrices variées issues de TIACs, donnant des résultats concordants avec les tests ELISA disponibles et les analyses PCR (Polymerase Chain Reaction). En parallèle, et de manière à mieux discriminer les variants de séquence, une quantification par approche top-down (analyse de protéine intacte) a été développée. L’analyse top-down permet une analyse globale de la séquence de l’ES ciblée à l’inverse du mode bottom up qui ne détecte que certains peptides. Une optimisation des paramètres SM et du traitement des données a été réalisée et un étalon interne a été sélectionné. Cela nous a permis d’obtenir, pour la SEA, une méthode de quantification absolue avec une sensibilité proche de la technique bottom-up. La méthode a finalement été confrontée à des analyses génomiques et ELISA lors de l’analyse de 13 souches productrices de variants de SEA. Enfin, nous avons souhaité élargir la quantification des ES au-delà des huit premières. Ainsi nous avons optimisé une méthode de préparation de l’échantillon sans anticorps. La méthode a été appliquée à des ES produites par des souches de S. aureus en milieu de culture. Nous avons pu identifier pour la première fois 24 types d’ES et plus de 162 variants. Des peptides spécifiques à chaque type et commun à l’ensemble des variants ont pu être identifiés. Par la suite, une méthode de quantification « label free » a été proposée, basée sur le principe « Hi3 » où le signal des trois peptides les plus intenses de chaque ES est exploité. A l’aide de cette méthode, nous avons pu identifier les ESs produites en forte quantité et établir leur cinétique de production par le S. aureus en milieu de culture, donnant de nouvelles indications sur les ES à rechercher en priorité lors d’épisodes de TIAC.

To be able to predict reversed phase liquid chromatographic (RPLC) retention times of contaminants is an asset in order to solve food contamination issues. The development of quantitative structure–retention relationship models (QSRR) requires selection of the best molecular descriptors and machine-learning algorithms. In the present work, two main approaches have been tested and compared, one based on an extensive literature review to select the best set of molecular descriptors (16), and a second with diverse strategies in order to select among 1545 molecular descriptors (MD), 16 MD. In both cases, a deep neural network (DNN) were optimized through a gridsearch., Pesticides; QSRR; Molecular descriptors; Deep neural network; Reversed-phase liquid chromatography; Selection of inputs.

The authors sincerely thank the other experts who participated in the working group set up by the French Agency for Food, Environmental and Occupational Health and Safety (ANSES) to prevent health risks associated with the consumption of shellfish contaminated with BTXs in France, that is to say: Katia Comte, Estelle Chaix, Nicolas Delcourt, César Mattei, Jordi Molgó and Raphaele Le Garrec.; International audience; In recent decades, more than 130 potentially toxic metabolites originating from dinoflagellate species belonging to the genus Karenia or metabolized by marine organisms have been described. These metabolites include the well-known and large group of brevetoxins (BTXs), responsible for foodborne neurotoxic shellfish poisoning (NSP) and airborne respiratory symptoms in humans. Karenia spp. also produce brevenal, brevisamide and metabolites belonging to the hemi-brevetoxin, brevisin, tamulamide, gymnocin, gymnodimine, brevisulcenal and brevisulcatic acid groups. In this review, we summarize the available knowledge in the literature since 1977 on these various identified metabolites, whether they are produced directly by the producer organisms or biotransformed in marine organisms. Their structures and physicochemical properties are presented and discussed. Among future avenues of research, we highlight the need for more toxin occurrence data with analytical techniques, which can specifically determine the analogs present in samples. New metabolites have yet to be fully described, especially the groups of metabolites discovered in the last two decades (e.g tamulamides). Lastly, this work clarifies the different nomenclatures used in the literature and should help to harmonize practices in the future.

International audience; The interest of studying organic plastic additives (OPAs) is growing since many of them are considered as toxic for human beings, marine organisms and have been found in the ecosystem. The impact and the extent of this pollution is not fully understood because of the lack of methods able to comprehensively analyze the wide diversity of OPAs. This study aims at offering a quick and efficient way to identify specific harmful plastic additives in polymer materials. For this purpose, a general method, for identifying 49 OPAs, has been successfully developed using pyrolyzer/thermal desorption-gas chromatography coupled to a high-resolution mass spectrometer (Py/TD-GC-HRMS). The retention indices were calculated for each compound and a high-resolution mass spectra database for plastic additives was implemented. This method was then applied to characterize the OPAs included in food containers that are frequently found in the environment. Four samples made of polypropylene (PP) and polylactic acid (PLA) coming from two distinct suppliers were then selected. A total of 10 additives, which were plasticizers (phthalates and adipates), antioxidants (bisphenol B) and phosphorous flames retardants, were found in these food packaging. Generally, higher peak signals were detected in the PP samples in comparison to PLA samples, except for tributyl phosphate and bisphenol B that were exclusively found in PLA samples. Moreover, the experimentations pointed out additives differences between suppliers, for products intended for the same use and made of the same polymer, for both PP and PLA samples highlighting the complexity of identifying and generalizing the additive content of plastic materials.

National audience; La pollution plastique est une menace pour l’environnement et les organismes. Elle les expose à différents dangers. Outre l’impact « physique » lié à l’ingestion de déchets plastique, l’impact « chimique », dû aux additifs plastiques ajoutés aux polymères au cours de leur formulation, est lui aussi problématique. L’utilisation de certains additifs est controversée du fait que certaines molécules se sont révélées être toxiques pour l’environnement, les organismes marins et l’Homme. De ce fait, l’analyse des additifs plastiques montre un intérêt grandissant.Afin d’avoir une meilleure compréhension de l’impact chimique de ces additifs, il est important de les identifier, et idéalement de les quantifier. Leur analyse dans les matrices polymériques est un challenge analytique pour différentes raisons : la diversité de nature chimique des additifs, leur présence généralement en mélange au sein des polymères, ainsi que les nombreuses étapes nécessaires pour la préparation des échantillons avec les approches plus « conventionnelles » telles que l’extraction au solvant. De ce fait, développer des méthodes analytiques relativement faciles et rapides présente un réel intérêt. Ainsi la pyrolyse couplée à la chromatographie gazeuse et la spectrométrie de masse haute résolution (Py-GC-HRMS) est un outil analytique qui permet une analyse relativement facile et rapide d’un échantillon. Cette technique permet de limiter voire de s’affranchir des étapes de préparation des échantillons, limitant alors leur potentielles contaminations avant l’analyse.Le but de cette étude est de développer une méthode en Py-GC-HRMS haute résolution pour l’identification et la quantification d’additifs plastiques organiques. Une méthode unique pour l’analyse de l'ensemble des additifs sélectionnés a été développée et a permis la constitution d’une base de donnée haute résolution contenant une large variété d’additifs plastiques. Cette méthode a ensuite été appliquée sur certains plastifiants dans le but de les quantifier. Elle montre son utilité pour la caractérisation et le dosage d’additifs dans différents échantillons de matrices polymériques.

National audience; En dix ans, les recherches sur les particules plastiques, nano- (NP) et microplastiques (MP), et leurs contaminants associés se sont largement développées. L’amélioration des connaissances de ces familles de contaminant s’étant accrue, les pratiques analytiques ont dû évoluer et être adaptées. La garantie de la qualité des résultats générés ne peut être obtenue que par la mise en commun des expertises, ainsi que la mise en place d’infrastructures et l’utilisation d’appareillages complémentaires.Dans le cadre du CPER Marco, différents laboratoires (Anses, LOG et ULCO) ont mis en place la plateforme 3PAC de façon à partager leurs équipements analytiques, méthodes, expertises et connaissances.En terme d’infrastructure, de nombreuses possibilités existent. Tout d’abord des systèmes d’aquarium avec un environnement contrôlé sont disponibles pour réaliser des études d’exposition. Une pièce « microplastique » permet de réaliser des manipulations (digestion, filtration) sous atmosphère maîtrisée et ainsi limiter les risques de contamination. La caractérisation des particules (taille, forme) peut être réalisée à l’aide de divers équipements : loupes binoculaires ou directement par microscopie via l’utilisation des capteurs optiques du µ-Raman ou du µ-FT-IR. Enfin, l’identification des particules ou des contaminants associés est rendue possible par l’utilisation de techniques complémentaires soit spectroscopiques (µ-Raman, µ-FT-IR), soit spectrométriques (Py-GC/MS et Py-GC/Orbitrap). L’acquisition de nouveaux équipements, complémentaires, est programmée de façon à venir enrichir les possibilités analytiques (microscopie confocale, technologie light scattering).Dès aujourd’hui, l’expertise des différents partenaires permet de couvrir un large champ de recherches allant des études en laboratoire, aux prélèvements sur le terrain. A titre d’exemple, des travaux récents ont porté sur l’étude de sédiments et organismes prélevés in situ, la réalisation d’études d’encagement d’organismes marins, ou encore l’exposition de bivalves en conditions contrôlées.Désormais, l’ambition de la plateforme 3PAC est de répondre aux appels à projets nationaux et internationaux portant sur l’étude des particules plastiques et de leurs conséquences. Ces projets seront menés au sein d’un écosystème analytique idoine, regroupant expertises, technologies et infrastructures.

International audience; Although relative air humidity (RH) strongly influences microbial survival, its use for fighting surface pathogens in the food industry has been inadequately considered. We asked whether RH control could destroy Listeria monocytogenes EGDe by envelope damage. The impact of dehydration in phosphate-buffered saline (PBS) at 75%, 68%, 43% and 11% RH on the bacterial envelope was investigated using flow cytometry and atomic force microscopy. Changes after rehydration in the protein secondary structure and peptidoglycan were investigated by infrared spectroscopy. Complementary cultivability measurements were performed by running dehydration–rehydration with combinations of NaCl (3–0.01%), distilled water, city water and PBS. The main results show that cell membrane permeability and cell envelope were greatly altered during dehydration in PBS at 68% RH followed by rapid rehydration. This damage led cells to recover only 67% of their initial volume after rehydration. Moreover, the most efficient way to destroy cells was dehydration and rehydration in city water. Our study indicates that rehydration of dried, sullied foods on surfaces may improve current cleaning procedures in the food industry.

To prevent health risks associated with the consumption of shellfish contaminated with BTXs in France, a working group was set up by the French Agency for Food, Environmental and Occupational Health & Safety (Anses). One of the aims of this working group was to propose a guidance level for the presence of BTXs in shellfish.; International audience; Brevetoxins (BTXs) are marine biotoxins responsible for neurotoxic shellfish poisoning (NSP) after ingestion of contaminated shellfish. NSP is characterized by neurological, gastrointestinal and/or cardiovascular symptoms. The main known producer of BTXs is the dinoflagellate Karenia brevis, but other microalgae are also suspected to synthesize BTX-like compounds. BTXs are currently not regulated in France and in Europe. In November 2018, they have been detected for the first time in France in mussels from a lagoon in the Corsica Island (Mediterranean Sea), as part of the network for monitoring the emergence of marine biotoxins in shellfish. To prevent health risks associated with the consumption of shellfish contaminated with BTXs in France, a working group was set up by the French Agency for Food, Environmental and Occupational Health & Safety (Anses). One of the aims of this working group was to propose a guidance level for the presence of BTXs in shellfish. Toxicological data were too limited to derive an acute oral reference dose (ARfD). Based on human case reports, we identified two lowest-observed-adverse-effect levels (LOAELs). A guidance level of 180 µg BTX-3 eq./kg shellfish meat is proposed, considering a protective default portion size of 400 g shellfish meat.

National audience; La pollution plastique est une menace pour l’environnement et les organismes. Elle les expose à différents dangers. Outre l’impact « physique » lié à l’ingestion de déchets plastique, l’impact chimique, dû aux additifs plastiques ajoutés aux polymères au cours de leur formulation, est lui aussi problématique. En effet, une fois dans l’environnement, les additifs plastiques peuvent être libérés de la matrice polymérique et diffuser dans l’eau environnante ou au sein des organismes après ingestion de particules de plastiques. L’utilisation de certains additifs est controversée car ils se sont révélés être toxiques pour l’environnement, les organismes marins ou l’Homme.Afin d’avoir une meilleure compréhension de l’impact de ces additifs sur les organismes, il est important de les identifier et idéalement de les quantifier. L’analyse des additifs plastiques est un challenge analytique pour différentes raisons. En comparaison avec les approches plus "traditionnelles", la pyrolyse couplée à une chromatographie gazeuse et spectrométrie de masse (Py-GC-MS) est un outil analytique qui permet une analyse relativement facile et rapide d’un échantillon. Cette technique permet une analyse quasi-directe des échantillons limitant alors les potentielles contaminations des échantillons lors des étapes de préparation précédant l’analyse.Le but de cette étude est de développer une méthode en Py-GC-MS pour l’identification et, si possible, la quantification d’additifs plastiques organiques. Après avoir sélectionné une soixantaine de ces additifs selon leur nocivité et utilisation (plastifiants, retardateurs de flammes, antioxydants, …), une méthode unique pour l’analyse de l'ensemble ces molécules a été développée et a permis la constitution d’une base de donnée haute résolution. Cette méthode sera appliquée sur divers échantillons pour identifier, voire quantifier, les additifs plastiques contenus dans plusieurs matrices polymériques. De plus, la mise en place d'une méthode de pyrolyse avec un gradient de température permet également de caractériser le type de polymère sur le même échantillon.

International audience; Many One Health surveillance systems have proven difficult to enforce and sustain, mainly because of the difficulty of implementing and upholding collaborative efforts for surveillance activities across stakeholders with different values, cultures and interests. We hypothesize that only the early engagement of stakeholders in the development of a One Health surveillance system can create an environment conducive to the emergence of collaborative solutions that are acceptable, accepted and therefore implemented in sustainable manner. To this end, we have designed a socio-technical framework to help stakeholders develop a common vision of their desired surveillance system and to forge the innovation pathway toward it. We implemented the framework in two case studies: the surveillance of antimicrobial resistance in Vietnam and that of Salmonella in France. The socio-technical framework is a participatory and iterative process that consists of four distinct steps implemented during a workshop series: (i) definition of the problem to be addressed, (ii) co-construction of a common representation of the current system, (iii) co-construction of the desired surveillance system, (iv) identification of changes and actions required to progress from the current situation to the desired situation. In both case studies, the process allowed surveillance stakeholders with different professional cultures and expectations regarding One Health surveillance to gain mutual understanding and to reconcile their different perspectives to design the pathway toward their common vision of a desired surveillance system. While the proposed framework is structured around four essential steps, its application can be tailored to the context. Workshop facilitation and representativeness of participants are key for the success of the process. While our approach lays the foundation for the further implementation of the desired One Health surveillance system, it provides no guarantee that the proposed actions will actually be implemented and bring about the required changes. The engagement of stakeholders in a participatory process must be sustained in order to ensure the implementation of co-constructed solutions and evaluate their effectiveness and impacts.

The European epidemic monophasic variant of Salmonella enterica serovar Typhimurium (S. 1,4,[5],12:i:-) characterized by the multi locus sequence type ST34 and the antimicrobial resistance ASSuT profile has become one of the most common serovars in Europe (EU) and the United States (US). In this study, we reconstructed the time-scaled phylogeny and evolution of this Salmonella in Europe. The epidemic S. 1,4,[5],12:i:- ST34 emerged in the 1980s by an acquisition of the Salmonella Genomic Island (SGI)-4 at the 3′ end of the phenylalanine phe tRNA locus conferring resistance to copper and arsenic toxicity. Subsequent integration of the Tn21 transposon into the fljAB locus gave resistance to mercury toxicity and several classes of antibiotics used in food-producing animals (ASSuT profile). The second step of the evolution occurred in the 1990s, with the integration of mTmV and mTmV-like prophages carrying the perC and/or sopE genes involved in the ability to reduce nitrates in intestinal contents and facilitate the disruption of the junctions of the host intestinal epithelial cells. Heavy metals are largely used as food supplements or pesticide for cultivation of seeds intended for animal feed so the expansion of the epidemic S. 1,4,[5],12:i:- ST34 was strongly related to the multiple-heavy metal resistance acquired by transposons, integrative and conjugative elements and facilitated by the escape until 2011 from the regulatory actions applied in the control of S. Typhimurium in Europe. The genomic plasticity of the epidemic S. 1,4,[5],12:i:- was demonstrated in our study by the analysis of the plasmidome. We were able to identify plasmids harboring genes mediating resistance to phenicols, colistin, and fluoroquinolone and also describe for the first time in six of the analyzed genomes the presence of two plasmids (pERR1744967-1 and pERR2174855-2) previously described only in strains of enterotoxigenic Escherichia coli and E. fergusonii.

En France, de nombreuses toxines non réglementées sont suivies dans le cadre du réseau de veille d’émergence des biotoxines marines dans les coquillages (EMERGTOX) dont le comité de pilotage est présidé par la DGAL et inclut la DGS, la Direction des pêches maritimes et del'aquaculture (DPMA), Santé publique France, l’Anses, l’Ifremer et la Direction de l’eau et de la biodiversité (DEB).Les brévétoxines sont incluses dans ce réseau depuis janvier 2018.Elles ont été mises en évidence pour la première fois en France en novembre 2018 dans des moules en Corse (117 μg/kg de glande digestive). Le prélèvement d’eau associé pour le suivi du phytoplancton a montré la présence de Karenia spp. (genre de microalgues dont certaines espèces produisent des brévétoxines). Il n’existe pas de limite maximale pour ces toxines dans le règlement (CE) n°853/2004. Les USA, l’Australie/Nouvelle-Zélande et le Mexique appliquent un seuil à 800 μg d'équivalent BTX-2/kg de chair de coquillages. Dans la norme CODEXSTAN 292–2008 (rev.2015) du Codex alimentarius, la limite maximale pour les brévétoxines est de 200 unités souris ou équivalent par kg de chair de mollusque vivant.Dans ce contexte, il a été demandé à l’Anses de fournir des éléments de réponse aux questions suivantes :Question 1. Quelles sont les données toxicologiques disponibles pour les brévétoxines ?Permettent-elles à l’Anses de proposer des valeurs toxicologiques de référence aiguë et chronique par voie orale ?Question 2. Sur la base des données toxicologiques identifiées par l’Anses, est-il possible de proposer une valeur guide dans les coquillages au-delà de laquelle des investigations complémentaires seraient demandées dans le cadre du réseau EMERGTOX ?Question 3. Quelles modalités de suivi des brévétoxines dans le milieu marin pourraient être recommandées dans le cadre du réseau EMERGTOX ? Quelles investigations complémentaires seraient à réaliser en cas de dépassement de la valeur guide ?Question 4. Compte tenu des données toxicologiques et au vu du contexte, existe-t-il une préoccupation de santé publique aux niveaux de contamination identifiés dans certaines zones marines françaises liée à la consommation de coquillages ?Question 5. Existe-t-il une préoccupation sanitaire liée à l’exposition aux brévétoxines par un contact direct avec de l’eau contaminée notamment en cas de baignade ou de pratique d’activités nautiques ou en cas d’exposition par inhalation à des embruns ?

This study was designed to optimise an analytical method for characterising TiO2 nanoparticles (NPs) in food additives and pharmaceuticals by inductively coupled plasma-mass spectrometry in single particle mode (spICP-MS). Several parameters, including transport efficiency (TE), were assessed and optimised using the NM-100 reference material. We found that self-aspiration for sample intake and use of the concentration-based method for TE was optimal for characterising TiO2 NPs. No spectral interference was observed with either 49Ti or 48Ti isotopes. The optimised Excel spreadsheet developed for this study not only provided additional parameters but gave results closer to the NM-100 reference value than the ICP-MS software. The method was then applied to the analysis of a selection of food samples and pharmaceuticals. The average diameter of TiO2 particles ranged from 86 to 179 nm in the food samples and from 131 to 197 nm in the pharmaceuticals, while the nanoparticular fraction was between 19 and 68% in food, and between 13 and 45% in pharmaceuticals.

Besides the infection properties in human and animals, S. aureus can produce different enterotoxins in food. The enterotoxins can cause vomiting and diarrhea, often involving many people., Currently, only 5 (SEA to SEE) out of 27 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 strains from different origins (food-poisoning outbreaks, human, and animal) were investigated by applying bioinformatics methods (phylogenetic analysis using the core genome and different alignments). SEG and SEI expression was tested in vitro using a sandwich enzyme-linked immunosorbent assay 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 Besides having infectious properties in human and animals, S. aureus can produce different enterotoxins in food. The 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, these 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.

National audience

International audience; In response to the massive use of biocides for controlling Listeria monocytogenes (hereafter Lm) contaminations along the food chain, strains showing biocide tolerance emerged. Here, accessory genomic elements were associated with biocide tolerance through pangenome-wide associations performed on 197 Lm strains from different lineages, ecological, geographical and temporal origins. Mobile elements, including prophage-related loci, the Tn6188_qacH transposon and pLMST6_emrC plasmid, were widespread across lineage I and II food strains and associated with tolerance to benzalkonium-chloride (BC), a quaternary ammonium compound (QAC) widely used in food processing. The pLMST6_emrC was also associated with tolerance to another QAC, the didecyldimethylammonium-chloride, displaying a pleiotropic effect. While no associations were detected for chemically reactive biocides (alcohols and chlorines), genes encoding for cell-surface proteins were associated with BC or polymeric biguanide tolerance. The latter was restricted to lineage I strains from animal and the environment. In conclusion, different genetic markers, with polygenic nature or not, appear to have driven the Lm adaptation to biocide, especially in food strains but also from animal and the environment. These markers could aid to monitor and predict the spread of biocide tolerant Lm genotypes across different ecological niches, finally reducing the risk of such strains in food industrial settings.

This study presents the development, validation and application of a new analytical approach for the simultaneous speciation analysis of Cr(III) and Cr(VI) in meat and dairy products by high-performance liquid chromatography (HPLC) coupled to inductively coupled plasma mass spectrometry (ICP-MS) and double spike species specific-isotope dilution (SS-ID). The species extraction was achieved by sequential complexation of Cr(III) with ethylenediaminetetraacetic acid (EDTA) and of Cr(VI) with 1,5-diphenylcarbazide (DPC) in the same analytical run. The HPLC separation of complexed species was carried out using a short (5 cm) microbore anion-exchange HPLC column and a mobile phase consisting of 0.01 mol L−1 HNO3 + 2.5% (v/v) MeOH + 0.30 mol L−1 EDTA (pH = 2) in isocratic elution mode with excellent baseline separation achieved in less than 3 min. The method was validated by means of the accuracy profile approach by carrying out 6 measurement series in duplicate on (six) different days over a timespan of two months. The quantification limit was 0.013 μg kg−1 for Cr(III) and 0.049 μg kg−1 for Cr(VI), respectively. The measurement bias corresponding to the validity domain ranged from 0.01 to 0.11%, whereas the coefficient of variation in terms of repeatability (CVr) varied from 2.9 to 11.6% (depending on the analyte level) for Cr(III) and from 6.7 to 11.8% for Cr(VI). Similarly, the coefficient of variation in terms of intermediate reproducibility (CVR) ranged from 6.8 to 13% for Cr(III) and from 6.8 to 25.9% for Cr(VI), respectively. The method was successfully applied to the analysis of a selection of food samples such as baby and semi-skimmed milk and steak beef samples. Cr(VI) was not quantified in any of these samples while Cr(III) levels ranged between 2.7 and 4.7 μg kg−1, which were comparable with the levels of total chromium analysed in the same samples by ICP-MS (accredited method). The method presented here with combined use of species specific isotope dilution and sequential species complexation is a powerful analytical tool for accurate and precise quantification of Cr(III) and Cr(VI) at trace levels and allows for correction of any species interconversion during sample preparation.

International audience

International audience; Among the enteric viruses implicated in foodborne outbreaks, the human norovirus and hepatitis viruses A and E (HAV and HEV) represent a serious public health concern. International standard ISO 15216 proposes methods for detecting HAV and norovirus (genogroups I and II) RNA from soft fruit, leaf, stem and bulb vegetables, bottled water or food surfaces. These methods had not previously been validated for detecting the targeted viruses in other foodstuffs such as multicomponent foods, nor for detecting other viruses in foodstuffs. The aim of this study was to characterise a method derived from the vegetable method described in ISO 15216 to detect HAV, HEV and norovirus in artificially-contaminated multicomponent foodstuffs according to the recent international standard ISO 16140-4. Results showed that the mean recovery rates for all settings did not differ according to the operator. The mean extraction yields ranged from 0.35% to 40.44% for HAV, 5.19% to 100% for HEV, 0.10% to 40.61% for norovirus GI and 0.88% to 69.16% for norovirus GII. The LOD(95) was 10(2) genome copies/g for HAV, HEV and norovirus GII and 10(3) genome copies/g for norovirus GI. The LOQ was 2.90 × 10(4), 1.40 × 10(3), 1.60 × 10(4) and 1.30 × 10(4) genome copies/g for HAV, HEV, norovirus GI and norovirus GII respectively. The MNV-1 process control was detected in 120 out of 128 RNA extracts analysed and was recovered with an efficiency of between 3.83% and 50.22%. The mean inhibition rates of quantitative real-time RT-PCR reaction ranged from 3.25% to 28.70% and varied significantly with the type of food matrix. The described method could be used to detect viruses in composite food products for routine diagnosis needs.

International audience; Bacillus thuringiensis or Bt is an ubiquitous bacteria used in agriculture for crop protection exploiting its insecticidal properties since the 1950s. At a taxonomic level, Bt belongs to the Bacillus cereus group (sensu lato) which also contains strains that may be involved in food quality and safety. While the use of commercial phytosanitary products based on Bt strains is an effective, easy-to-use and low cost treatment for crop protection, the persistence of spores on treated vegetables is not always compatible with food microbiological criteria. The BtID project carried out (1) experimental characterizations on the diversity of B. thuringiensis and the positioning of commercial strains within the B. cereus Group; (2) a review of agricultural practices for the protection of Bt-based plants as well as (3) the development of methodologies and decision-making tools to identify, distinguish and trace the Bt strains authorized at the European level, from field to fork. This collection of commercial strains made it possible to sequence the genome of three distinct strains, which represent the majority of strains used in Bt biopesticides, and to achieve a detailed characterization of the behavior of these highly specialized strains, as well as their occurrence on raw materials (vegetables) and processed food.; Bacillus thuringiensis ou Bt est une bactérie ubiquiste utilisée dans le domaine agricole en protection des cultures pour ses propriétés insecticides depuis les années 1950. Au niveau taxonomique, Bt appartient au groupe Bacillus cereus (sensu lato), qui contient également des souches pouvant être impliquées dans la qualité et sécurité des aliments. Même si l’utilisation de produits phytosanitaires à base de souches commerciales de Bt est un moyen efficace, facile d’utilisation et avantageux pour la protection des cultures, la persistance de spores sur les végétaux traités n’est pas toujours compatible avec les critères microbiologiques fixés pour leur utilisation dans les denrées alimentaires. Le projet BtID a permis (1) d’évaluer la diversité de B. thuringiensis et le positionnement de souches commerciales au sein du groupe B. cereus ; (2) de passer en revue les pratiques agricoles pour la protection des plantes à base de Bt ainsi que (3) de développer des méthodologies et outils d’aide à la décision permettant d’identifier, distinguer et tracer les souches de Bt autorisées au niveau européen, du champ à l’assiette. La collecte de ces souches commerciales a permis le séquençage du génome de trois représentants distincts majoritaires utilisés en biocontrôle, une caractérisation fine du comportement de ces souches hautement spécialisées, ainsi que d’étudier leur prévalence au niveau des matières premières (légumes) et produits finis (alimentaires transformés).

International audience; Bacillus thuringiensis or Bt is an ubiquitous bacteria used in agriculture for crop protection exploiting its insecticidal properties since the 1950s. At a taxonomic level, Bt belongs to the Bacillus cereus group (sensu lato) which also contains strains that may be involved in food quality and safety. While the use of commercial phytosanitary products based on Bt strains is an effective, easy-to-use and low cost treatment for crop protection, the persistence of spores on treated vegetables is not always compatible with food microbiological criteria. The BtID project carried out (1) experimental characterizations on the diversity of B. thuringiensis and the positioning of commercial strains within the B. cereus Group; (2) a review of agricultural practices for the protection of Bt-based plants as well as (3) the development of methodologies and decision-making tools to identify, distinguish and trace the Bt strains authorized at the European level, from field to fork. This collection of commercial strains made it possible to sequence the genome of three distinct strains, which represent the majority of strains used in Bt biopesticides, and to achieve a detailed characterization of the behavior of these highly specialized strains, as well as their occurrence on raw materials (vegetables) and processed food.; Bacillus thuringiensis ou Bt est une bactérie ubiquiste utilisée dans le domaine agricole en protection des cultures pour ses propriétés insecticides depuis les années 1950. Au niveau taxonomique, Bt appartient au groupe Bacillus cereus (sensu lato), qui contient également des souches pouvant être impliquées dans la qualité et sécurité des aliments. Même si l’utilisation de produits phytosanitaires à base de souches commerciales de Bt est un moyen efficace, facile d’utilisation et avantageux pour la protection des cultures, la persistance de spores sur les végétaux traités n’est pas toujours compatible avec les critères microbiologiques fixés pour leur utilisation dans les denrées alimentaires. Le projet BtID a permis (1) d’évaluer la diversité de B. thuringiensis et le positionnement de souches commerciales au sein du groupe B. cereus ; (2) de passer en revue les pratiques agricoles pour la protection des plantes à base de Bt ainsi que (3) de développer des méthodologies et outils d’aide à la décision permettant d’identifier, distinguer et tracer les souches de Bt autorisées au niveau européen, du champ à l’assiette. La collecte de ces souches commerciales a permis le séquençage du génome de trois représentants distincts majoritaires utilisés en biocontrôle, une caractérisation fine du comportement de ces souches hautement spécialisées, ainsi que d’étudier leur prévalence au niveau des matières premières (légumes) et produits finis (alimentaires transformés).

International audience; The aim of this work was to test a surveillance protocol able to detect extended-spectrum β-lactamase (ESBL)-, cephalosporinase (AmpC)- and carbapenemase (CP)-producing gram-negative bacteria in three conveniently chosen dairy farms with known prior occurrences of ESBL- and CP-producing strains. The protocol was applied monthly for a year. At each visit, 10 healthy lactating dairy cows were rectally swabbed, and raw milk filters (RMFs) were sampled in two of the three farms. Bacterial isolation was based on a first screening step with MacConkey agar supplemented with 1 mg/L cefotaxime and commercial carbapenem-supplemented media. We failed to detect CP-producing strains but showed that ESBL- Escherichia strains, found in one farm only (13 strains), were closely associated with multi-drug resistance (12 out of 13). The limited number of conveniently selected farms and the fact that RMFs could not be retrieved from one of them limit the validity of our findings. Still, our results illustrate that ESBL-status changes monthly based on fecal swabs and negative herds should be qualified as “unsuspected” as proposed by previous authors. Although surveillance of farm statuses based on RMF analysis could theoretically allow for a better sensitivity than individual swabs, we failed to illustrate it as both farms where RMFs could be retrieved were constantly negative. Determination of CP herd-level status based on RMFs and our surveillance protocol was hindered by the presence of intrinsically resistant bacteria or strains cumulating multiple non-CP resistance mechanisms which means our protocol is not specific enough for routine monitoring of CP in dairy farms.

International audience; Bacillus thuringiensis (Bt) belongs to the Bacillus cereus (Bc) group, well known as an etiological agent of foodborne outbreaks (FBOs). Bt distinguishes itself from other Bc by its ability to synthesize insecticidal crystals. However, the search for these crystals is not routinely performed in food safety or clinical investigation, and the actual involvement of Bt in the occurrence of FBOs is not known. In the present study, we reveal that Bt was detected in the context of 49 FBOs declared in France between 2007 and 2017. In 19 of these FBOs, Bt was the only microorganism detected, making it the most likely causal agent. Searching for its putative origin of contamination, we noticed that more than 50% of Bt isolates were collected from dishes containing raw vegetables, in particular tomatoes (48%). Moreover, the genomic characterization of isolates showed that most FBO-associated Bt isolates exhibited a quantified genomic proximity to Bt strains, used as biopesticides, especially those from subspecies aizawai and kurstaki. Taken together, these results strengthen the hypothesis of an agricultural origin for the Bt contamination and call for further investigations on Bt pesticides.

International audience; Analysis of organic plastic additives (OPAs) associated to plastic polymers is growing. The current review outlines the characteristics and the development of (multi-step) pyrolysis coupled with a gas chromatography mass spectrometer (Py-GC/MS) for the identification and semi-quantification of OPAs. Compared to traditional methods, Py-GC/MS offers advantages like suppressing extensive steps of preparation, limiting contamination due to solvents and the possibility to analyse minute particles. Its key advantage is the successive analysis of OPAs and the polymeric matrix of the same sample. Based on the studied articles, numerous methods have been described allowing identification and, in some case, semi-quantification of OPAs. There is nevertheless no gold standard method, especially given the huge diversity of OPAs and the risks of interferences with polymers or other additives, but, among other parameters, a consensus temperature seems to arise from studies. More broadly, this review also explores many aspects on the sample preparation like weight and size of particles and calibration strategies. After studying the various works, some development prospects emerge and it appears that methodological developments should focus on better characterizing the limits of the methods in order to consider which OPAs can be quantified and in which polymers this is feasible.