Journal Article Comparison of the virucidal efficacy of peracetic acid, potassium monopersulphate and sodium hypochlorite on bacteriophages P001 and MS2 Get access T. Morin, T. Morin French Agency for Food Environmental and Occupational Health & Safety Ploufragan‐Plouzané Laboratory Viral Fish Pathology Unit Université Européenne de Bretagne Technopôle Brest Iroise Plouzané FranceACTALIA Sécurité des Aliments Villers Bocage France Correspondence Thierry Morin, French Agency for Food, Environmental and Occupational Health & Safety ‐ Ploufragan‐Plouzané Laboratory, Viral Fish Pathology Unit, Technopôle Brest‐Iroise ‐ BP70 ‐ 29 280 Plouzané ‐ France. E‐mail: thierry.morin@anses.fr Search for other works by this author on: Oxford Academic Google Scholar H. Martin, H. Martin French Agency for Food, Environmental and Occupational Health & Safety Fougères Laboratory, Cedex France Search for other works by this author on: Oxford Academic Google Scholar C. Soumet, C. Soumet French Agency for Food, Environmental and Occupational Health & Safety Fougères Laboratory, Cedex France Search for other works by this author on: Oxford Academic Google Scholar R. Fresnel, R. Fresnel French Agency for Food, Environmental and Occupational Health & Safety Fougères Laboratory, Cedex France Search for other works by this author on: Oxford Academic Google Scholar S. Lamaudière, S. Lamaudière French Agency for Food, Environmental and Occupational Health & Safety Fougères Laboratory, Cedex France Search for other works by this author on: Oxford Academic Google Scholar A.L. Le Sauvage, A.L. Le Sauvage ACTALIA Sécurité des Aliments Villers Bocage France Search for other works by this author on: Oxford Academic Google Scholar K. Deleurme, K. Deleurme French Agency for Food, Environmental and Occupational Health & Safety Fougères Laboratory, Cedex France Search for other works by this author on: Oxford Academic Google Scholar P. Maris P. Maris French Agency for Food, Environmental and Occupational Health & Safety Fougères Laboratory, Cedex France Search for other works by this author on: Oxford Academic Google Scholar Journal of Applied Microbiology, Volume 119, Issue 3, 1 September 2015, Pages 655–665, https://doi.org/10.1111/jam.12870 Published: 01 September 2015 Article history Received: 19 March 2015 Revision received: 15 May 2015 Accepted: 29 May 2015 Published: 01 September 2015
Introduction Chemical (eg pesticides, veterinary drugs, etc.) and bacteriological contaminants (eg. foodborne pathogens) could contaminate animal and plant derived food products for human consumption. Some antibiotic residues (eg. chloramphenicol, nitrofuran metabolites, dyes) are banned in foodstuffs of animal origin (eg. milk, meat, eggs, etc.) in European Union because of toxicological risks for the consumer. The European Regulation has set Minimum Required Performance Limits (MRPL) [1] or Reference Point for Action (RPA) for banned substances [2]. Food containing residues of substances at or above the MRPL or RPA are declared non-compliant and consignments are rejected from the consumer’s market. Screening methods are the first stage of food control and so are essential for food safety monitoring. Conventional screening methods are microbiological methods (eg. plate tests, tube tests), immunological methods (eg. ELISA, radioimmunoassays) or physico-chemical methods (Thin Layer Chromatography, High Performance Liquid Chromatography (HPLC), liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS)). These methods sometimes lack of sensitivity or specificity; they also could be time and money consuming. There is thus a need to develop novel screening methods for antibiotic residues detection, preferably with the potential for the field-testing (eg. farm control, self-control). Electrochemical biosensors make it possible to develop a promising and economically interesting approach. Electrochemical immunosensor An innovative electrochemical method based on disposable Screen Printed Carbon Electrodes (SPCE), coupled to magnetic beads (MB), allowing the simultaneous detection of 3 families of antibiotics in milk, was published by a Spanish academic team [3]. This technique presents major advantages: low cost (eg. disposable electrodes, potentiostat), promising detection limits, portability, and possible automatisation. Our laboratory has evaluated the transferability of the method. An electrochemical immunosensor has been developed for the detection of chloramphenicol residues in milk as a proof of concept. The matrix effect (milk samples) was high and so sample preparation has to be improved to reduce matrix effects. The objective is to develop an amperometric bead-based immunosensor for the multiplex detection of banned antibiotics (eg. chloramphenicol, nitrofuran metabolites, dyes) in bovine milk. Method Antibodies (Abs) against antibiotics are grafted on the surface of magnetic beads (MBs). Milk samples and antibiotic conjugated with Horseradish peroxidase (HRP) are mixed with MBs-Abs. A competition occurs between the HRP conjugates and the antibiotic residues if present in the sample, for the binding to the antibody. The MBs are washed to remove free antibiotics and conjugates. Then, a Screen Printed Carbon Electrode (SPCE) with MBs on its surface (maintained by a magnet) is soaked into a buffer solution containing hydroquinone; when adding hydrogene peroxide (H 2 O 2 ) to the solution, an amperometric signal is produced, due to the enzymatic activity of HRP and measured. The amperometric signal is inversely proportional to the antibiotic concentration in the sample. Results and Conclusions Screening methods for the detection of veterinary drugs in food products have to be validated according to the European regulation [1] and to the European guideline for the validation of screening methods [4]. After the development and the optimization of the analytical parameters (eg. sample preparation, HRP concentration, incubation time, applied potential, etc), the methods developed for single compounds will be evaluated and validated according to the European regulations. Then the single compound methods will be merged into one multiplex method if possible. The results will be presented to the conference, discussing the advantage sand drawbacks of amperometric biosensors for the screening of antibiotic residues in food products. References 1. Commission Decision (EC) N° 2002/657 of 12 August 2002 implementing Council Directive 96/23/EC concerning the performance of analytical methods and interpretation of results. 2002: Official Journal of European Communities. p. 8-36. 2. Commission Regulation (EC) N° 470/2009 laying down Community procedures for the establishment of residue limits of pharmacologically active substances in foodstuffs of animal origin 2009, The European parliement and the Council: Official Journal of the European Union p. 11-22. 3. Conzuelo F, Ruiz-Valdepeñas Montiel V, Campuzano S, Gamella M, Torrente-Rodríguez RM, Reviejo AJ, Pingarrón JM. 2014. Rapid screening of multiple antibiotic residues in milk using disposable amperometric magnetosensors. Anal. Chim. Acta. 820:32-38. 4. CRL, Guideline for the validation of screening methods for residues of veterinary medicines (initial validation and transfer). 2010: Available from:<http://ec.europa.eu/food/food/chemicalsafety/residues/lab_analysis_en.htm>: Guideline_Validation_Screening_en.pdf. p. 1-18.
The Infiniplex for milk® (IPM) kit is a quick method for the simultaneous and qualitative detection of more than 100 molecules including antibiotic residues, mycotoxins, anti-inflammatories and antiparasitic drugs into a single test that does not require milk treatment.The IPM® kit was validated according to the European decision EC/2002/657 and according to the European guideline for the validation of screening methods (2010). Our validation was focused only on antibiotic residues. The washing step was identified as the most critical step of the assay. Insufficient washes could cause a significant background noise that prevents imaging. Positive controls have to be freshly prepared each day (insufficient stability).The method was specific with a low false-positive rate of 1.7% on 5 discrete test regions (DTR) ((beta-lactams, lincomycin, virginiamycin, quinolones and sulphonamides)) and a false-positive rate of 0% on the 26 other DTR. During our validation, the 42 determined detection capabilities CCβ for 12 antibiotic families (aminoglycosides, cephalosporins, lincosamides, macrolides, miscellaneous antibiotics, penicillins, phenolated polymixins, polypeptide antibiotics, quinolones, sulphonamides, tetracyclines) were at between once and twice the decision levels stated by the manufacturer. Forty CCβ determined were lower than the respective regulatory limits (i.e. MRL, RC, MRPL) in milk, except for tilmicosin (1.5 times the MRL) and neospiramycin (>1.25 times the MRL). The estimated CCβ of thiamphenicol, cloxacillin, danofloxacin, sulphathiazol, ceftiofur and sulphamonomethoxine were lower than or at the MRL. However, it was difficult to approach an accurate CCβ with only qualitative results. It is impossible to know whether or not we were close to the cut-off value. The software could be improved by differentiating between low-positive and high-positive results. The results of our participation in three qualitative proficiency tests in 2016 and 2017 for the detection of quinolones, tetracyclines and sulphonamides in cows' milk were very satisfactory.
Efficient screening methods are needed to control antibiotic residues in eggs. A microbiological kit (Explorer® 2.0 test (Zeu Inmunotech, Spain)) and an immunobiosensor kit (Microarray II (AM® II) on Evidence Investigator™ system (Randox, UK)) have been evaluated and validated for screening of antibiotic residues in eggs, according to the European decision EC/2002/657 and to the European guideline for the validation of screening methods. The e-reader™ system, a new automatic incubator/reading system, was coupled to the Explorer 2.0 test. The AM II kit can detect residues of six different families of antibiotics in different matrices including eggs. For both tests, a different liquid/liquid extraction of eggs had to be developed. Specificities of the Explorer 2.0 and AM II kit were equal to 8% and 0% respectively. The detection capabilities were determined for 19 antibiotics, with representatives from different families, for Explorer 2.0 and 12 antibiotics for the AM II kit. For the nine antibiotics having a maximum residue limit (MRL) in eggs, the detection capabilities CCβ of Explorer 2.0 were below the MRL for four antibiotics, equal to the MRL for two antibiotics and between 1 and 1.5 MRLs for the three remaining antibiotics (tetracyclines). For the antibiotics from other families, the detection capabilities were low for beta-lactams and sulfonamides and satisfactory for dihydrostreptomycin (DHS) and fluoroquinolones, which are usually difficult to detect with microbiological tests. The CCβ values of the AM II kit were much lower than the respective MRLs for three detected antibiotics (tetracycline, oxytetracycline, tylosin). Concerning the nine other antibiotics, the detection capabilities determined were low. The highest CCβ was obtained for streptomycin (100 µg kg-1).
Presentation de l'entreprise ...2 I) Introduction 3 II) Synthese bibliographique .4 II) 1.Escherichia coli (E. coli) ........4 II) 2. Les Antibiotiques II) 2. a Generalites ......4 II) 2. b La resistance aux Cephalosporines de 3eme generation (C3G) 5 II) 3. Les biocides II) 3. a Generalites .......6 II) 3. b Caracteristiques des biocides etudies 7 II) 3. c La tolerance aux biocides etudies 8 II) 4. Relation entre tolerances aux biocides et resistances aux antibiotiques II) 3. a Contexte general ........9 II) 3. b Lien entre resistance aux antibiotiques et tolerance aux biocides etudies 10 III) Materiels et Methodes ...11 III) 1. Les souches bacteriennes ...11 III) 2. Biocides et Antibiotiques ...11 III) 3. Test de sensibilite aux biocides et aux antibiotiques (phase 1) III) 3. a Mesure de la Concentration Minimale Inhibitrice (CMI) des souche aux biocides .....12 III) 3. b Mesure de la CMI des souches aux antibiotiques ..12 III) 4. Adaptation des souches aux biocides (phase 2) 13 III) 5. Mesure de la Concentration Minimale Bactericide (CMB) (phase 3) 13 III) 6. Analyses statistiques .........14 IV) Resultats 15 IV) 1. Determination de la sensibilite des souches aux biocides et aux antibiotiques IV) 1. a Choix des souches .15 IV) 1. b Sensibilite des souches aux trois biocides etudies (BC, Chx et Hex) 15 IV) 1. c Sensibilite des souches aux antibiotiques 16 IV) 2. Adaptation des souches au chlorure de benzalkonium, a la chlorhexidine et a l'hexamidine IV) 2. a Impact de l'adaptation sur la sensibilite au biocide concerne 17 IV) 2. b Impact de l'adaptation a l'un des trois biocides sur la sensibilite croisee aux autres biocides 19 IV) 2. c Impact de l'adaptation au chlorure de benzalkonium sur la sensibilite aux antibiotiques 19 IV) 3. Determination de la concentration minimale bactericide...22 V) Discussion 24 VI) Conclusion et Perspectives ......27 Bibliographie 28 Annexes 31
Polymerase chain reaction (PCR) was used after a short pre-enrichment culture to detect Salmonella subspecies in chicken fillets. A direct PCR assay performed with chicken meat inoculated with Salmonella Typhimurium produced no PCR products. Six different DNA extraction protocols were tested to recover efficiently Salmonella DNA after a short incubation period. Three of them gave results that were reliable, rapid and sensitive. Successful protocols used Proteinase K and/or a centrifugation step to concentrate the samples. For reliable detection of Salmonella subspecies, a few thousand bacterial cells per ml must be present. To obtain this number of bacterial cells with an inoculation of about one cell in 25 g of ionized food products, it was necessary to incubate samples for at least 10 h before PCR. A larger inoculum of approximately 10 cells in 25 g of ionized food products, required 8 h in culture broth to give positive results by PCR-based assay.
