Recherche de résidus d'antibiotiques dans la filière bovine française: des progrès à intensifier

International audiencePour la recherche de résidus antibiotiques dans la viande bovine, par exemple, des méthodes de chromatographie liquide couplée à la spectrométrie sont désormais validées et utilisées en France, permettant de détecter rapidement plus de 60 antibiotiques. Sont ciblées les carcasses “anormales” ou qui font l’objet d’informations suspectes sur le document d’accompagnement.La filière bovine française a obtenu des résultats très encourageants sur les échantillons de 2013 (les résultats 2016 sont en cours d’analyse). Le non-respect des temps d’attente ou de la prescription, chez les vaches de réforme et les veaux, est principalement en cause pour les résidus dans la viande bovine. Des progrès semblent encore possibles

Development of a multi-class method to determine nitroimidazoles, nitrofurans, pharmacologically active dyes and chloramphenicol in aquaculture products by liquid chromatography-tandem mass spectrometry

International audienceLC-MS/MS method was developed for the efficient identification and quantification of 21 banned substances including various nitroimidazoles, nitrofurans, pharmacologically-active dyes and chloramphenicol, respectively in aquaculture products. The sample preparation was started by acid-treatment with 2-nitrobenzaldehyde (NBA) to liberate matrix-bound residues of nitrofurans. A modified QuEChERS method was optimized for the extraction and clean-up of the target analytes. The metabolites of the four conventional nitrofurans (nitrofurantoin, furazolidone, nitrofurazone and furaltadone) and of three other nitrofurans (nifursol, nifuroxazide, and nitrovin), and an underivatizable nitrofuran (nifurpirinol) were simultaneously detected. Furthermore, 21 banned substances were quantified by LC-MS/MS with ESI using one single injection. To evaluate and validate the performance of the method the criteria of the Decision (EC) no 2002/657 were applied. Decision limit (CCα) of target analytes ranged 0.067-1.655 μg/kg in aquaculture products. The recovery ranged 77.2%-125.6%, and the relative standard deviations of inter-day analyses (RSD) were less than 25%

Development of a liquid chromatography-tandem mass spectrometry method to determine colistin, bacitracin and virginiamycin M1 at cross-contamination levels in animal feed

International audienceCross-contamination of animal feed with antibiotics may occur during manufacturing in feed mills, because shared production lines can be used for medicated and non-medicated feed, but may also occur during transport, storage and at the farm level. This is a major issue in the current context where antimicrobial usage must be controlled in order to maintain their effectiveness. A LC-MS/MS method was developed for the determination of colistin, bacitracin A and virginiamycin M1 in feed for pigs, poultry and rabbits at concentrations similar to those encountered in cross-contamination. After investigating various issues related to colistin behaviour and matrix effects, we successfully validated this method according to the requirements of European regulations in terms of linearity, trueness, precision, limit of quantification and limit of decision. Trueness ranged 88.6–107.8% and precision ranged 12.6–21.2%. We then applied this method to the analysis of medicated pig feed to check the performance of the method on “real” samples of medicated feed. We subsequently analysed non-medicated pig, and rabbit feed samples, collected directly on farms, to check the rate of cross-contamination. No samples were contaminated by colistin, bacitracin, or virginiamycin

Liquid chromatography–tandem mass spectrometry method for the analysis of N -(3-aminopropyl)- N -dodecylpropane-1,3-diamine, a biocidal disinfectant, in dairy products

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Metabolism of the Marine Phycotoxin PTX-2 and Its Effects on Hepatic Xenobiotic Metabolism: Activation of Nuclear Receptors and Modulation of the Phase I Cytochrome P450

PTX-2 is a marine biotoxin frequently found in shellfish that can lead to food intoxication in humans. Information regarding PTX-2 metabolism is scarce, and little is known of its effect on xenobiotic-metabolizing enzymes (XME) or its molecular pathways. The aim of this study was consequently to examine PTX-2 Phase I metabolism using rat and human liver S9 fractions, and also to assess the capability of PTX-2: (i) to modulate the gene expression of a panel of Phase I (CYP450) and II (UGT, SULT, NAT, and GST) enzymes, as well as the Phase III or 0 (ABC and SLCO) transporters in the human hepatic HepaRG cell line using qPCR; (ii) to induce specific CYP450 in HepaRG cells measured by immunolabeling detection and the measurement of the cells’ activities; and (iii) to activate nuclear receptors and induce CYP promoter activities in HEK-T and HepG2 transfected cell lines using transactivation and reporter gene assay, respectively. Our results indicate that PTX-2 hydroxylation occurred with both rat and human S9 fractions. Whereas PTX-2 mostly upregulated the gene expression of CYP1A1 and 1A2, no induction of these two CYP activities was observed. Lastly, PTX-2 did not act as an agonist of CAR or PXR. Due to its effects on some key XME, more attention should be paid to possible drug–drug interactions with phycotoxins, especially as shellfish can accumulate several phycotoxins as well as other kinds of contaminants

Antimicrobial Resistance in the Bacterial Communities of the Rainbow-Trout Filet

National audienceThe role of food in the routes of transmission of resistant bacteria and antimicrobial resistance genes (ARG) is yet to be explored. Farmed fish filets can carry antibiotic-resistant bacteria because of their environmental exposure (animal farms, human activities, aquatic environment…). The presence of such bacteria on foodstuff must be evaluated as it might present a risk for the consumers. To extend our knowledge about the farmed fish filet resistome, ARG were sought in the bacterial communities of fresh rainbow-trout filets. Antimicrobial residues were also dosed in the filets. The analyses were performed on fishes originating from a single batch sampled in two conditions: a) filets excised in laboratory conditions (N = 14) and b) filets excised in an industrial facility (N = 14). Each filet was divided, one sample was rinsed and the DNA was extracted to perform the ARG investigation and one sample was analysed to perform the antimicrobial residues dosing. Insights about events occurring during the lifetime of the fishes, such as diseases, treatments, vaccines and pond cleaning frequency were provided by the breeders. The ARG were detected and quantified using a 245 primers pairs set. The set were chosen after bibliography analyses [1] and in silico verification. It was designed to detect resistances to 10 antimicrobial classes, including beta lactams, macrolides, tetracyclines, aminoglycosides, phenicols, as well as multidrug efflux pumps and biocides resistance genes. The amplification was realised thanks to the Smartchip Real-Time PCR technology (Takara). The residues of 75 antimicrobials, covering 10 antimicrobial classes, were looked for and quantified by LC-MS/MS. Some ARG were detected at Ct around 25 (tetL, tetB, sul1), as well as biocide resistance genes (qacEΔ1). Oxytetracycline residues were detected in 13 filets (out of 28) in concentrations lower than the authorized maximum residue limit. The concentrations ranged from 5,84 to 40,2 μg/kg and the authorized maximum residue limit is 100 μg/kg in the fish muscle. Fishes were treated with oxytetracycline more than 6 months before the sampling. Overall, few antimicrobial resistance genes were detected and the detected genes displayed high Ct, suggesting that this genes had low prevalence in the bacterial communities. Presence of tetracycline resistance genes and oxytetracycline residues have been observed in fish samples from a batch where an oxytetracycline treatment has been applied. No correlation has been established yet. Further investigations are necessary in order to understand the links between the presence of residues and genes, and the phenotypic resistance

A non-targeted LC-HRMS approach for detecting exposure to illegal veterinary treatments: The case of cephalosporins in commercial laying Hens

International audienceCephalosporins are of particular importance in human medicine and should be reserved for second-line curative treatment in the veterinary field to avoid any emerging antimicrobial resistance. Due to misuse of ceftiofur in the poultry sector in France, it is now recommended to completely stop using cephalosporins in this sector. Methods currently used for the control of veterinary practices are mostly based on liquid chromatography coupled to mass spectrometry in a targeted mode, including parent compounds and any major metabolites. The aim of the present study was to evaluate the relevance of untargeted metabolomic approaches to highlight a possible exposure of laying hens to cephalosporins using a predictive model including selected treatment biomarkers. An experimentation carried out on living animals involved the administration of cefquinome and ceftiofur. Three biological matrices—droppings, eggs and liver—were investigated. Metabolites were extracted and analysed by liquid chromatography coupled to high resolution mass spectrometry in a full scan mode. Metabolites impacted by the treatment were selected by using univariate and multivariate statistical analyses. Predictive models built from the potential biomarkers selected in the "droppings" matrix were validated and able to classify “treated” and “control” hens. PLS-DA and logistic regression models were compared and both models gave satisfactory results in terms of prediction. Results were of less interest for other matrices in which only biomarkers of exposure to cefquinome were detected

Tissue distribution, metabolism, and elimination of Victoria Pure Blue BO in rainbow trout: Main metabolite as an appropriate residue marker

International audienceVictoria Pure Blue BO is a dye that bears some therapeutic activity and that can be retrieved in effluent or may be used in aquaculture as a prohibited drug. In this study, the metabolism and tissue distribution during uptake and depuration of VPBO were investigated in order to propose a residue marker of illegal treatment in fish. The dye was administered to rainbow trout (oncorhynchus mykiss) for one day by water bath at a dose of 0.1 mg.L(-1). The concentrations of VPBO in all tissues increased rapidly during the treatment period, reaching a C(max) of 567 ± 301 μg.L(-1) in plasma and 1846 μg kg(-1) ±517 for liver after 2 h. After placing the rainbow trout in a clean water bath for a 64 day-period of depuration, the concentrations in the tissues and plasma decreased to reach comparable levels for muscle and for skin after 33 days. The concentrations measured were still above the LOQ at 2.26 ± 0.48 μg kg(-1) for muscle and 2.85 ± 1.99 μg kg(-1) for skin at the end of the depuration period. The results indicated the existence of 14 phase I metabolites and one glucuronide conjugated metabolite. Non-compartmental analysis was applied to assess the pharmacokinetic parameters. The half-life in edible muscle of the main metabolite detected, deethyl-leuco-VPBO, was found to be 22.5 days compared to a half-life of 19.7 days for the parent VPBO. This study provides new information to predict a VPBO drug treatment of aquacultured species via a proposed new residue marker

Metabolism of the lipophilic phycotoxin 13-Desmethylspirolide C using human and rat in vitro liver models

International audience13-Desmethylspirolide C (13-SPX-C) is a phycotoxin produced by dinoflagellates which can accumulate in shellfish. 13-SPX-C induces neurotoxic effects in rodents through blockade of nicotinic acetylcholine receptors. As no human intoxication has been to date attributed to the consumption of 13-SPX-C-contaminated seafood, this toxin is not regulated according to the Codex Alimentarius. Nevertheless, shellfish consumers can be exposed to 13-SPX-C via shellfish consumption. In order to follow the fate of the toxin after ingestion and to verify whether metabolic detoxification could explain the lack of human intoxications, we assessed the metabolism of 13-SPX-C using several in vitro liver systems. First, both phase I and II reactions occurring with rat and human liver S9 fractions were screened. Our results indicated that 13-SPX-C was almost completely metabolized with both rat and human liver S9. Using a receptor binding assay towards nicotinic acetylcholine receptors we demonstrated that the resulting metabolites showed less affinity towards nicotinic acetylcholine receptors than 13-SPX-C. Finally, we showed that 13-SPX-C induced a pronounced increase of gene expression of the drug-metabolizing enzyme cytochrome P450 (CYP) CYP1A2. The role of this CYP in 13-SPX-C metabolism was clarified using an innovative in vitro tool, CYP1A2-Silensomes™. In summary, this study highlights that liver first-pass metabolism can contribute to the detoxification of 13-SPX-C