Research activities of the Fougères Laboratory

PROMETHEE is the proteomics and metabolomics facility of ANSES. It is located at the ANSES Fougères laboratory. Its mission is to provide expertise and support to ANSES entities in carrying out their projects. The platform also has its own research activity focused on technical and methodological developments aimed at characterising the modes of action of biological and chemical contaminants, their impact on health and their fate. The platform uses a TimsTOF Pro2 mass spectrometer coupled to a nano-UHPLC nanoelute2 system (Bruker). The acquisition of this instrument was financially supported by ANSES, the Regional Council of Brittany and the EU through FEDER funding.

Development of tools to monitor the species involved in ciguatera fish poisoning in the Caribbean Sea

Funding: French Development Agency

This project will work to develop, in Caribbean countries, protocols for monitoring the species involved in ciguatera fish poisoning. It will use environmental screening via quantitative PCR (qPCR) and will assess the toxicity of the dinoflagellate species Gambierdiscus, Fukuyoa and Coolia found along a north-south gradient in Central America and the Caribbean islands. The project will provide participants from various countries with training where they will acquire skills relating to culturing, biomass growth and the preparation of samples for qPCR analysis to help them identify the algal compounds responsible for ciguatera fish poisoning in the Caribbean. The Fougères Laboratory will perform cell tests (in particular the Neuro-2A test) to assess the cytotoxicity of dinoflagellate extracts.

Funding: ANSES-INRAE thesis (Biocare) and ANR-JCJC (Baobab)

The Biocare and Baobab projects aim to better understand the role of biocides, which are applied daily to guarantee safety at all stages of the food chain, in the development of antimicrobial resistance. The adaptation of microbial flora to the constraints imposed by environmental factors in the food industry is a major public health concern that is associated in particular with the increase in antimicrobial resistance phenomena. These projects intend, initially, to study the emergence of antibiotic-resistant variants in biofilms treated with biocides. They will do so using a collection of E. coli strains that have been isolated in the food chain over the past 20 years as part of the annual surveillance plans of the National Reference Laboratory for antimicrobial resistance. These variants will then be phenotypically (minimum inhibitory concentration depending on the antibiotic/biocide, growth rates, biofilms) and genotypically (whole genome sequencing (WGS), comparative genomics) characterised in order to identify the molecular markers underlying this adaptation. The ability of the variants to recolonise various materials representative of those used in the food chain (stainless steel, polytetrafluoroethylene, etc.) will also be investigated, as will their survival within microbial consortia of increasing complexity, using strains isolated from the food industry. These results will help better assess the risks related to the use of biocides in a context of antimicrobial resistance and its spread, and identify the genetic determinants involved in these adaptive processes.

Temperature-responsive nanogels for targeted delivery of microRNAs in wound healing and tissue regeneration applications

Funding: Horizon 2020 programme

This project’s objective is to develop an innovative nanogel, containing iron oxide nanoparticles and targeted microRNAs, within a polymer for skin treatments and tissue regeneration. Iron oxide promotes the induction of fibrogenesis and microRNAs are involved in modulating the expression of specific genes. Various nanogels will be synthesised. Their toxicity and genotoxicity will be evaluated in vivo and in vitro.

Development of electrochemical and colorimetric biosensors for the detection of disinfectant biocide residues in the dairy industry

Funding: Brittany Region – Sustainable Attractiveness Strategy regional aid

Optical biosensors are used to detect a wide variety of food contaminants because they have the best characteristics (sensitivity, speed, low cost, high throughput) for field use. Already evaluated as part of the laboratory’s reference missions for the detection of antimicrobial residues, they could be a promising alternative for the detection of biocide residues. This detection could rely on rinses from operations to clean and disinfect food contact surfaces. The Capbiota project is funded by the Brittany Region as part of a Sustainable Attractiveness Strategy call to recruit a PhD researcher for 18 months. It will deal with the development, optimisation, evaluation and validation of optical (colorimetric and fluorimetric) and electrochemical biosensors, for the detection of quaternary ammonium compounds and amines in the dairy industry. In addition to drawing from the unit's expertise relating to the detection of antimicrobial and veterinary medicinal product residues, the project will benefit from the scientific and technical skills of a laboratory of the University of Perpignan (Biosensors - Analysis - Environment (BAE)).

Innovative materials including silver ions to guarantee the safety of aquaculture products

Funding: European Maritime and Fisheries Fund (EMFF)

The Silverprotect project is led by the Bacteriology and Parasitology of Fishery and Aquaculture Products (B3PA) Unit of ANSES's Laboratory for Food Safety. Its aim is to adopt an innovative approach by focusing on the prevention of biofilms and by acting upstream. It intends to use polymer surface coatings (for transport crates, conveyor belts, walls, ceilings, etc.) including silver ions as antibacterial agents supplementing cleaning and disinfection operations. The technical and hygienic performance of these antimicrobial materials will be evaluated in collaboration with the PureZone consortium, the University Institute of Technology (IUT) in Saint-Brieuc, the Technical Centre for Agri-Food Expertise in Saint-Lô, and industry stakeholders. This project will enable the French aquaculture sector to be competitive and will also help it reduce its environmental footprint and manage food safety. The Antibiotics, Biocides, Residues and Resistance (AB2R) Unit is involved in the laboratory study on the synergistic or antagonistic effects of silver ions in antibacterial materials in combination with the biocides used in hygiene procedures. This study also has a second component: the evaluation of bacterial adaptation and resistance following exposure to antimicrobial products.

Combined phenotypic and genomic approach to improve knowledge of the persistence of Listeria monocytogenes in aquaculture

Funding: European Maritime and Fisheries Fund (EMFF)

This project is being carried out in the context of an overall increase in the number of listeriosis cases over the past several years, both in Europe and around the world. Several outbreaks of Listeria monocytogenes (Lm) infections involving smoked or marinated salmon have recently been detected, consolidating this pathogenic bacterium’s position as a major challenge for the aquaculture processing sector. Several campaigns have demonstrated that Lm can persist for long periods in shellfish cooking and smoked salmon production facilities. Foods can then become contaminated during their production, in particular at the processing stage. Other studies have revealed the presence of viable but non-culturable pathogenic strains on surfaces; it is difficult to detect these strains and validate the actual effectiveness of cleaning and disinfection procedures. This project’s primary objectives are to genomically characterise (typing – WGS) persistent Lm isolates in the cooked shrimp & shellfish and smoked salmon & trout sectors, and identify the microbiological characteristics of the persistent isolates that would explain their increased ability to survive in the environment. This characterisation will combine genomic and phenotypic approaches. The AB2R Unit is involved particularly in phenotypic characterisation, with the identification of decontamination methods effective against persistent strains in conditions close to real industrial conditions.

Optimising and reducing the use of disinfectant products in the poultry and fish farming sectors

Funding: Special Fund for Agricultural and Rural Development (CASDAR)

This project's objectives are to optimise the use of disinfectant biocides throughout the poultry (from hatchery to slaughterhouse) and fish production chains, in order to maintain their effectiveness and reduce practices that pose risks in terms of worker and consumer health and ecotoxicity.

The AB2R Unit, in collaboration with the Actalia technical centre, is responsible for evaluating optimised practices as well as methods that supplement the use of chemical disinfectants (enzymatic detergents, barrier flora) to improve and reduce the use of disinfectant biocides. This work is focusing on situations of major concern identified during surveys undertaken at the start of the project. The AB2R Unit is carrying out effectiveness tests for these optimised methods in standardised laboratory conditions, for the bacterial indicators most frequently encountered at the various steps of each sector’s production chain. Actalia will then test the most advantageous methods in a technology centre, in conditions as close as possible to field conditions.

Food contamination by antibiotics 

Funding: Brittany Region and ANSES

The cross-contamination of animal feed by antibiotics, resulting from the use of the same production lines for all types of feed, including medicated feedingstuffs, was demonstrated by surveillance plans implemented on French farms in 2017 and 2018. A thesis under way is assessing the risk of residues being transferred to animal feed and also the risk of resistance emerging in the microbiota of pigs given feed contaminated by the antibiotics most commonly found during surveillance plans. These results will help generate useful data for the European Food Safety Authority (EFSA), to supplement its risk assessment and the calculation of maximum levels of cross-contamination related to the use of medicated feedingstuffs.

Science-based risk governance of nano-technology

Funding: Europe (H2020, NMBP-13)

RiskGONE aims to provide solid procedures for consistent governance of risks related to engineered nanomaterials. The project's partners are working to develop new tools or modify existing ones to identify with greater certainty the environmental and human health impacts of a number of nanomaterials. These tools and the results of tests using them will then be integrated into the work of a European Risk Governance Council (ERGC), a group of individuals with different areas of expertise on nanomaterials that is tasked with making governance decisions on the safety of specific materials. A risk governance framework will be developed to address nanomaterial safety governance in a coherent and scientifically robust way.

Find out more on the project’s website.

Quaternary ammonium compounds – bowel-intestinal-microbiotal

Funding: National Research Programme for Environmental and Occupational Health

Preliminary exploratory study investigating the impact of quaternary ammoniums on permeability and the microbiota, consequences for chronic inflammatory bowel diseases. For the first time, the interplay between exposure to low concentrations of disinfectant biocides, the gut microbiota and the intestinal barrier will be studied to expand knowledge on chronic inflammatory diseases using a holistic interdisciplinary approach. This project is coordinated by the Infinite unit (Inserm - University of Lille - Lille University Hospital) and involves three units of the Fougères Laboratory (ARC, EMAD, AB2R).

High-throughput genetic toxicology platform using a 3D HepaRG human hepatic cell model for the screening of genotoxic compounds

Funding: Brittany Region

The assessment of the toxicity of chemical compounds is still partly based on studies undertaken in animals, where the harmful effects observed at high doses are extrapolated to low doses in humans. These approaches are very expensive and time-consuming, use a large number of animals and provide toxicity data that are not very robust and can only partially be extrapolated to humans. Moreover, they are not suitable for assessing the thousands of substances to which humans are currently exposed. Toxicology is therefore faced with the challenge of developing in vitro models that are as predictive as possible of human toxicity and are also suitable for high-throughput screening.

The PlaTox3D project is building on the laboratory’s previous work with its proposal to design a high-throughput genetic toxicology platform for the 3D culture of HepaRG liver cells. Using a high-content screening cell imaging tool, it intends to conduct a high-throughput analysis of DNA damage based on the CometChip system, developed by Trevigen, which is particularly well suited to spheroids. The ultimate goal will be to validate the use of this genetic toxicology platform for the HepaRG spheroid model by studying a list of compounds of interest (both negative and positive) in order to assess the sensitivity, specificity and predictivity of the method used. This platform may be used with other cell types from various organs such as the kidneys, brain or digestive tract, in order to study organ-specific toxicity using both 3D and conventional 2D models.

Use of New Approach Methodologies for the hazard assessment of nanofibers. Lot 1: nanocellulose oral exposure: gastrointestinal digestion, nanofiber uptake and local effects.

Funding: European Food Safety Authority (EFSA)

The aim of this project is to design and conduct a set of new approach methodology (NAM) studies to address gaps in data on the hazards associated with nanocellulose (NC), a nanomaterial found in novel foods and food supplements. The results may be included in the regulatory hazard assessment of nanocellulose for consumers exposed via food. The first part will involve a battery of in vitro tests to better understand the hazards associated with NC and its mode of action on cells and determine whether a relationship can be established between its toxicity and its physico-chemical characteristics. The results of this first part will be used to select two or three materials that will be studied more thoroughly. This study will focus on the digestion or degradation of NC by the human microbiome, the assessment of uptake and potential crossing of the intestinal barrier, and the evaluation of the local effects, including inflammation and genotoxicity, of NC on the intestinal epithelium.

Materials having the most adverse effects or whose physico-chemical properties are likely to be associated with increased hazards will be subject to level 3 trials that will assess repeated-dose toxicity. A proper dispersion protocol will be developed and an in-depth physicochemical characterisation of each material will be performed in addition to transmission electron microscopy imaging of NC in situ, i.e. in cells and in colon samples.

Indicators of infectious risks in bathing water

Funding: Consortium for Research, Higher Education and Innovation in New Caledonia (CRESICA)

The health authorities of the three Provinces of New Caledonia are regularly faced with complaints from bathers and coastal users (foul odours, headaches, sore throats, nausea and burning skin and eyes) when sea water turns green, pink or brown. However, the observed effects are not systematic and sometimes, certain effects such as dermatoses are reported without the sea water changing colour. To protect the population, the authorities order beach closures without knowing exactly when the beaches will be reopened. Furthermore, they do not have access to infectious risk indicators to protect the population when the water is not an unusual colour.

This project aims to define such indicators. Phytoplankton samples will be collected from various places at different times of the year. To determine the level of toxicity of the samples, tests with cultured mammalian cells will be performed in the Fougères Laboratory.

​​​​​​An integrative test system strategy for the identification of endocrine disruptors inducing metabolic disorders

Funding: Europe (H2020, BHC-27)

The objective of OBERON is to develop new strategies for the identification of endocrine disruptors by developing, improving and validating a battery of tests. To better understand the complex biology of these hazards and predict their effects in humans, the project will combine, in an integrative framework, various test systems and experimental methods (in vitro in human cells and tissue, and in vivo in zebrafish) with metabolomic, lipidomic, transcriptomic and computational modelling methods (in silico and systems biology). The aim of this interdisciplinary project is to better evaluate endocrine disruptors through the use of mechanistic approaches to toxicity. It will provide validated alternative risk assessment methods in partnership with national and European regulatory agencies.

Find out more on the project’s website.

Prevention and mitigation of mycotoxin contamination of food and feed caused by climate change

Funding: Europe (Interreg Atlantic Area)

The main objective of Agritox is to provide the Atlantic Area food and feed industries with information and technical solutions to avoid contamination by mycotoxins, which is an increasingly relevant issue due to the influence of climate change.  Through training and the dissemination of information, the project's partners engage with stakeholders to promote awareness of mycotoxins, help identify methods for detecting existing mycotoxins, and facilitate technology transfer. The project will identify mycotoxin risks for food and feed and will develop a comprehensive reference database of mycotoxins that will provide Atlantic Area stakeholders with occurrence information and technical support. The Agritox project will also develop a cost-efficient, easy-to-use Mycotoxin Alert System, which will be tested for different food and feed sectors and different stages of the production life cycle.

Find out more on the project’s website.

Monitoring the risk of food poisoning associated with the use of Bt bio-insecticides

Funding: ANSES

Bacillus thuringiensis (Bt) is number one on the global market of microbial bio-insecticides and populations can be exposed to it via food. However, it belongs to the same bacterial group as Bacillus cereus, which has been associated with several food poisoning outbreaks in humans. The aim of this project is to generate a better understanding of the risks related to the use of bio-insecticides containing Bacillus thuringiensis.

Pilot study on new methodological approaches for the risk assessment of tebufenpyrad, Part 1: Development of a physiologically based kinetic model, coupled with pulmonary and topical exposure.

Funding: European Food Safety Authority (EFSA)

The objectives of this project are to estimate the fate of the active substance tebufenpyrad, used as a pesticide, in the human body and evaluate its role in the development of Parkinson's disease. Part 1 of the project aims to estimate internal concentrations in various compartments of the human body. This part is complementary to Part 2, which will study the toxic effects on mitochondria. The two parts combined will ultimately allow a conclusion to be drawn as to the risk of mitochondrial complex I inhibition and the occurrence of Parkinson's syndrome associated with this inhibition.

State-Region Plan Contract – Infectio

Funding: European Regional Development Fund
Partners: University of Rennes 1 and the ANSES Ploufragan-Plouzané-Niort Laboratory

The CPER-Infectio-1 project has enabled the acquisition of specific equipment and led to a better structuring between the project partners. It has enhanced the activities of the Fougères Laboratory, particularly with regard to host/pathogen relationships and the study of the internal and external exposome, because the funding has increased the laboratory's ability to carry out biological analyses and assess the effects of chemical contaminants.

The equipment acquired has provided new impetus for the research topics studied by ANSES and the community of infectious disease specialists in Brittany. It concerns:

• an automatic spiral plater, used for the enumeration of bacteria in samples with fewer intermediate dilutions;
• a flow cytometer for cell counting;
• an automated stage microscope, for reading stained tissue on slides. This system uses fluorescence and can carry out measurements automatically with little intervention by the operator.

Quantitative assessment of variability in Salmonella excretion, and impact of biocide use on the development of antimicrobial resistance after cleaning/disinfection, on pig farms

Funding: ANSES

This project was launched to address two concerns in the pig sector: bacterial resistance to antibiotics and food safety. Coordinated by the AB2R Unit, it also involved ANSES's Ploufragan-Plouzané-Niort Laboratory (HQPAP, EPISABE Units) and Regulated Products Assessment Department (U2EB Unit). The results showed that biocidal products containing quaternary ammoniums and glutaraldehyde applied during cleaning and disinfection operations led to an approximate 1,000-fold reduction in total flora and the quantity of E. coli. Exposure to these biocides did not cause E. coli to develop resistance to four target antibiotics or to biocidal substances representative of the main chemical classes used in this sector. The level of Salmonella excretion by slaughter pigs was relatively low with two-thirds of samples having less than 10 Salmonella per gram of faeces.

Adaptive traits of Listeria monocytogenes (Lm) to its diverse ecological niches

Funding: European One Health EJP Programme

Along with Salmonella and Shiga toxin-producing Escherichia coli (STEC), Listeria monocytogenes (Lm), which causes listeriosis, is one of the main causal agents involved in foodborne infections in the European Union, in terms of disease severity and mortality rates. However, the ecology of Lm is still poorly understood and the capacity of some strains to adapt to the environmental conditions encountered in the food industry makes the production of safe, high-quality food a major challenge. The ListAdapt project aimed to decipher the molecular mechanisms of the adaptation of Lm to its various ecological niches by comparing both genotypic and phenotypic data from a large and balanced set of strains from the environment, animals, foods and clinical cases in several European countries. A cutting-edge technology based on WGS was used to identify new key factors underlying the adaptation of Lm to various environments, in order to obtain new information from these factors regarding useful markers for the detection of Listeria monocytogenes.

Reports and presentations relating to this work are available on the website of the One Health EJP. 

Improving phenotypic antimicrobial resistance testing 

Funding: European One Health EJP Programme

As part of Work package 1 of the IMPART project, the AB2R Unit was in charge of developing, improving and validating a screening method for the detection, characterisation and confirmation of colistin resistance in Enterobacteriaceae sampled from animals on farms and from their meat at distribution. The unit organised several ring tests, in which the project’s 11 European partners participated, to evaluate the method developed and also the method proposed as part of the second work package, for the detection of carbapenemase-producing Enterobacteriaceae. The analysis of results showed that further tests are necessary to improve the performance of the methods. Reports and presentations relating to this work are available on the website of the One Health EJP and on the Zenodo platform.

Funding: European Commission

As part of its reference missions, the laboratory evaluated and validated the technical performance of several enzyme-linked immunosorbent assay (ELISA) kits. Three of the four tested kits for the detection of residues of nitrofuran metabolites (prohibited substances) in aquaculture products (shrimp, fish) showed detection capabilities below the current European regulatory requirements (1 µg/kg) with a false-positive rate of around 10%. For colistin, an antibiotic primarily used in pigs and poultry to treat gastrointestinal infections that is included on the list of highest priority critically important antimicrobials of the World Health Organization, three kits were evaluated and validated. They were all highly specific (no false positives) for the detection of colistin in pig and poultry muscle. However, only one of the kits showed a truly satisfactory detection capability (30 µg/kg) in relation to the maximum residue limit (150 µg/kg). These findings were highlighted in publications in European peer-reviewed journals.

Pre-validation of endocrine disruptor characterisation methods

Funding: Pepper platform

ANSES is participating in the pre-validation of two tests aimed at detecting substances with endocrine disrupting properties: the hPlacenTox-PE test and LC-MS/MS profiling in the H295R steroidogenesis assay. These two tests were selected by the Pepper platform’s Relevance Committee, which brings together private and public partners conducting research into endocrine disruptors. The project will initially work to verify the conditions in which methods can be transferred to laboratories; it will then try them out on test substances.

Find out more on the project’s website.

found in milk, in several species (cows, sheep and goats) and with several antibiotics

Funding: Nouvelle-Aquitaine Region and ANSES 

This project concerns the evaluation of antimicrobial residues in milk. The presence of residues promotes the emergence and development of resistant bacteria. However, the studies undertaken face various challenges, especially from an analytical standpoint. They are long, complex and expensive. They could benefit from recent progress in the field of physiologically based pharmacokinetic (PBPK) modelling. This project, conducted in collaboration with internal (Ploufragan-Plouzané-Niort Laboratory) and external (Inserm, University of Poitiers, the company Ceva Santé Animale, and the agricultural high schools of Melle and Venours) partners, focused on three milk-producing animal species. Residue levels in milk are currently being analysed.

Contamination of feathers and byproducts by antibiotic residues on poultry farms: characterisation of exposure levels and risks to the food chain and the environment.

Funding: EcoAntibio plan

The objective of the ECOPLUME project is to study the contamination of feathers and byproducts by antibiotic residues on poultry farms. This project therefore intends to assess the occurrence of antibiotic residues in feathers from farm animals and evaluate levels of exposure via the reuse of these feathers. Since poultry feathers in flocks treated with antibiotics can be contaminated by faecal material containing antibiotic-resistant bacteria, studying the change in the percentage of resistant bacteria in chicken droppings and feathers following antimicrobial treatment will help assess the impact of using feathers in feed for farmed fish and as fertiliser on the environmental spread of antimicrobial resistance.

Funding: French National Research Agency (ANR)

The ANR Ocean-15 project, which had four partners and was coordinated by the National University of Ireland, Galway, lasted five years. It strove to better understand the behaviour of a toxic microalga, Ostreopsis cf. ovata, found in the Mediterranean.  It also identified the compounds produced and their role in the toxic effects observed in humans. Ostreopsis secretes toxins called ovatoxins that are analogues of a well-known toxin known as palytoxin. Effects reported in humans involving the skin, eyes, ears, nose and larynx have been associated with Ostreopsis blooms. Although these toxins are also found in some seafood products, no cases of human poisoning related to their consumption have been reported. The project's participants purified two variants (ovatoxins a and d) from Ostreopsis cultures. The Fougères Laboratory tested their toxicity using various cell models. Both ovatoxins were found to be toxic to cells of the nervous system and to lung cells, with no major differences between the variants and at levels similar to those observed with palytoxin. However, in human cultures mimicking the intestinal barrier, both toxins were less toxic than palytoxin.