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A novel and reliable method to quantify residual levels of N-(3-aminopropyl)-N-dodecylpropane-1,3-diamine in dairy products using ion-pairing reversed-phase liquid chromatography–tandem mass spectrometry (LC–MS/MS) was developed and fully validated. Sample extraction was done with salting-out technique using acetonitrile and sodium chloride. For LC–MS/MS, the analyte was detected using positive electrospray ionization (ESI+) and two multiple reaction monitoring (MRM) transitions were monitored. The method was validated in the 5–150 µg kg−1 range using total error approach. Thus, performance criteria of the method were evaluated. Relative standard deviations for trueness and precision were lower than 10%; with the exception of hard pressed cheese at 5 µg kg−1 for precision. The limit of quantification (LOQ) was around 5–7 µg kg−1 depending on the matrix of interest. The method was successfully applied to accurately quantify N-(3-aminopropyl)-N-dodecylpropane-1,3-diamine in 146 various dairy products with a maximum contamination level of 225 µg kg−1 in cheese.

Aims Our aim was to assess the effects of step-wise exposure to didecyl dimethyl ammonium chloride (DDAC) on the antimicrobial (antibiotics and biocides) susceptibilities of food-associated bacterial strains. Methods and Results Adaptive responses of bacterial strains were investigated by exposing the strains daily to increasing sub-inhibitory concentrations of DDAC for 7 days. Following adaptation to DDAC, a 3-fold increase in the MIC values for this biocide was observed in 48% of the Escherichia coli and Listeria monocytogenes strains, and 3% of the Salmonella strains. Reduced susceptibility to other biocides was found with the most important increase in MIC for benzalkonium chloride (BC) and a commercial biocide formulation (Galox Horizon) containing DDAC and glutaraldehyde, for all species except Salmonella. Increase in antibiotic MIC values was more pronounced in Escherichia coli in terms of antibiotic numbers and of magnitude (from 4- to 32-fold increase) and, to a lesser extent, in Salmonella strains. Most of these strains had acquired resistance to ampicillin, cefotaxime, ceftazidime, chloramphenicol and ciprofloxacin. Conclusions The effects of exposure to DDAC on biocides and antibiotics susceptibilities depend upon the bacteria species. Significance and Impact of the Study Extensive use of DDAC at sub-inhibitory concentrations may lead to the development of antibiotic-resistant bacteria and may represent a public health issue This article is protected by copyright. All rights reserved.

Aims The phagicidal activity of peroxy products against the virulent bacteriophage P001 infecting lactic acid bacteria and bacteriophage MS2 used as a surrogate of enteric viruses (EVs) was evaluated and compared to sodium hypochlorite using the EN 13610 European suspension test and a surface test developed in our laboratories. Methods and results Infectivity tests were adapted and/or developed to determine the activity of disinfectants against reference P001 phage of Lactoccocus lactis and F-specific RNA phage MS2 of Escherichia coli in conditions simulating practical use. Similar concentrations of sodium hypochlorite were phagicidal against both bacteriophages, either at 0·05-0·125% of active chlorine using the suspension test or at 0·12-0·5% using the surface test. For Potassium monopersulphate (MPS), phagicidal concentrations varied from 0·006 to 0·012% whatever the type of test and phages. However, for peracetic acid products (PAP) used in suspension, concentrations 55 times higher were necessary against MS2 (0·271%) than against P001 (0·005%). With the surface test, 0·089-0·178% concentrations of PAP were effective against MS2, but these concentrations were 16-32 times greater than needed against P001. Conclusions Sodium hypochlorite and MPS had similar phagicidal activities against P001 and MS2, but PAP did not. Significance and impact of the study This is the first comparative study to investigate through suspension and surface tests the difference in resistance to peroxy compounds between a reference bacteriophage (P001) used to evaluate phagicidal concentrations in European standards and a surrogate of EVs (MS2). Results underline the importance of validation tests on pertinent surrogates of viruses or bacteriophages to adjust the concentration of disinfectants for use in the food and water industries.

International audience; Quaternary ammonium compounds (QACs) are both cationic surfactants and biocidal substances widely used as disinfectants in the food industry. A sensitive and reliable method for the analysis of benzalkonium chlorides (BACs) and dialkyldimethylammonium chlorides (DDACs) has been developed that enables the simultaneous quantitative determination of ten quaternary ammonium residues in dairy products below the provisional maximum residue level (MRL), set at 0.1mgkg(-1). To the best of our knowledge, this method could be the one applicable to milk and to three major processed milk products selected, namely processed or hard pressed cheeses, and whole milk powder. The method comprises solvent extraction using a mixture of acetonitrile and ethyl acetate, without any further clean-up. Analyses were performed by liquid chromatography coupled with electrospray tandem mass spectrometry detection (LC-ESI-MS/MS) operating in positive mode. A C18 analytical column was used for chromatographic separation, with a mobile phase composed of acetonitrile and water both containing 0.3% formic acid; and methanol in the gradient mode. Five deuterated internal standards were added to obtain the most accurate quantification. Extraction recoveries were satisfactory and no matrix effects were observed. The method was validated using the total error approach in accordance with the NF V03-110 standard in order to characterize the trueness, repeatability, intermediate precision and analytical limits within the range of 5-150mugkg(-1) for all matrices. These performance criteria, calculated by e.noval((R)) 3.0 software, were satisfactory and in full accordance with the proposed provisional MRL and with the recommendations in the European Union SANTE/11945/2015 regulatory guidelines. The limit of detection (LOD) was low (

AIMS: The virucidal activity of peroxy-products was evaluated and compared with sodium hypochlorite using the EN 14675 European suspension test and a surface test developed in our laboratory. The classical approach on infectivity of viruses was complemented with a prospective approach on virus genomes. METHODS AND RESULTS: Both infectivity tests were adapted and/or developed to determine the activity of disinfectants against reference bovine enterovirus type 1 [enteric cytopathogenic bovine orphan virus (ECBO)] and resistant hepatitis A virus (HAV) in conditions simulating practical use. Similar concentrations of active chlorine were virucidal against both viruses, either at 0*062% using the suspension test or at 0*50-1% using the surface test. However, for potassium monopersulfate and peracetic acid products, concentrations of approximately three times (3%) to 72 times (9%) higher were necessary against HAV than ECBO when determined with the suspension test. With the surface test, 4-8% peroxy-products were virucidal against HAV, either 16 times more peroxy-products concentrations than against ECBO. No significant impact on the targeted area of the viral genome measured by real-time RT-PCRs was obtained for ECBO and HAV suspensions treated with disinfectants, even with doses higher than the minimal virucidal concentrations. CONCLUSIONS: Sodium hypochlorite, but not peroxy-products, had similar activity against ECBO and HAV. No relation could be established between infectivity tests and genome destruction. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first comparative study that investigates with novel suspension and surface tests the reduction of infectivity and genome destruction of two resistant viruses by peroxy-compounds. The results and conclusions collected with European standards are discussed.

In recent years, raw fruits and vegetables have frequently been involved in foodborne transmission to humans of enteric viruses, particularly noroviruses and hepatitis A virus (HAV). Although viral contamination can occur during all steps of food processing, primary production is a critical stage on which prevention measures must be focused to minimize the risk of infection to consumers. Postharvest sanitation may be a valid technological solution for decreasing the bacterial load on fresh raw material, but there is a lack of data concerning the effectiveness of this process on enteric viruses. In this study, we compared the survival of two human norovirus surrogates, the feline calicivirus (FCV), and the murine norovirus (MNV-1), and of HAV on lettuce after water washing with bubbles and with or without ultrasound, and washing with bubbles in the presence of active chlorine (15 ppm) or peroxyacetic acid-based disinfectant (100 ppm). Cell culture and quantitative RT-PCR assays were used to detect and quantify the viruses on the surface of the lettuce after the sanitizing treatments. Levels of viral inactivation on the lettuce leaves were not significantly different between washing with bubbles and washing with bubbles plus ultrasound and were not dependant on the quantification method. A simple washing without disinfectant resulted in a decrease of approximately 0.7 log units in the quantity of virus detected for HAV and FCV and of 1.0 log unit for MNV-1. In the experimental set-up including a washing step (with or without ultrasound) followed by washing for 2 min in the presence of disinfectants, 15 ppm of active chlorine was found more effective for inactivating FCV (2.9 log units) than HAV and MNV-1 (1.9 log units and 1.4 log units, respectively) whereas 100 ppm of peroxyacetic-based biocide was found effective for inactivating FCV (3.2 log units) and MNV-1 (2.3 log units), but not HAV (0.7 log units). Quantitative RT-PCR results indicated that the presence of viral RNA did not correlate with the presence of infectious viruses on disinfected lettuce, except for MNV-1 processed with chlorine (15 ppm). In comparison with water washing, a substantial additional decrease of genomic FCV titer (1.1 log units) but no significant reduction of the genomic titers of HAV and MNV-1 were found on lettuce treated with chlorine (15 ppm). No significant effect of the disinfection step of lettuce with peroxyacetic-based biocide (100 ppm peracetic acid) was found by qRT-PCR on all genomic viral titers tested. This study illustrates the necessity of determining the effectiveness of technological processes against enteric viruses, using a relevant reference such as HAV, in order to reduce the risk of hepatitis and gastroenteritis by exposure to vegetables.

Campylobacters are a primary cause of human bacterial enteritis worldwide. They are usually considered susceptible to the disinfectant molecules used in the food industry. The purpose of this study was to see if campylobacters could survive cleaning and disinfection in poultry slaughterhouses and whether the strains recovered could contaminate carcasses during processing. Samples obtained from the environment before and after cleaning and disinfection (transport crates, processing equipment surfaces, scald tank water) and from birds (fresh droppings, neck skins) were collected during 7 investigations in 4 different slaughterhouses. Out of 41 samples collected, 30 Campylobacter jejuni strains were recovered from the surfaces of processing equipment before cleaning and disinfection procedures in three slaughterhouses and 9 C. jejuni out of 51 samples collected were found after cleaning. The study was then focused on one slaughterhouse to trace passage of the pathogen on poultry carcasses. The antimicrobial resistance phenotypes (P) (minimum inhibitory concentration, MIC) of the C. jejuni isolates collected in this slaughterhouse were determined. Nine phenotypes could be distinguished. Three of these were of interest as they were found in isolates recovered after cleaning and disinfection procedures. The genotypes (G) were determined by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) of isolates with one of the three phenotypes of interest. Clusters constructed by combining the phenotype and genotyping observations (P ⁎ G type) were compared between isolates obtained after cleaning and disinfection, and isolates from droppings, neck skin and transport crate samples of slaughtered poultry flocks. Only one P ⁎ G type of strain was recovered from surfaces after cleaning and disinfection and from neck skin samples but was also recovered from transport crates. Our findings indicate that C. jejuni is able to survive overnight on food processing equipment surfaces, after cleaning and disinfection procedures, and that these strains may contaminate carcasses during the slaughter process. These results add to our understanding of poultry carcass contamination and highlight the need to develop ways of reducing the risk of human infection with Campylobacter through the consumption of poultry products.

The use of chloramphenicol in veterinary medicine was banned in the EU in 1994. As the Community Reference Laboratory for antibiotic residues in food of animal origin, one of our functions is to organize inter-laboratory studies. A first inter-laboratory study for the analysis of chloramphenicol (CAP) in milk by ELISA kits was organized in 2001 and a second one for the detection of CAP in pig muscle by ELISA kits in 2002. These studies were intended to allow participants to control their CAP ELISA methods when used routinely and also to compare the performances of various ELISA kits for the detection of chloramphenicol in milk (commercial or in-house kits). In 2001, 15 participants received ten randomly coded frozen milk samples (four blank samples and six spiked milk samples from 0.5 to 5.0 μg/l). In 2002, 20 participants received eight randomly coded frozen muscle samples (two blank samples and six incurred muscle samples from 2.1 to 6.5 μg/kg). They were asked to analyse each sample in triplicate with ...

A collaborative study involving 14 laboratories was conducted to determine residues of ampicillin in porcine muscle tissue by using a liquid chromatographic method developed for multipenicillin analysis that can quantitate 8 penicillin compounds (benzylpenicillin, phenoxymethylpenicillin, ampicillin, amoxicillin, nafcillin, oxacillin, cloxacillin, and dicloxacillin) at trace levels in muscle tissue. This method involves extraction of the penicillins with phosphate buffer, pH 9, followed cleanup and concentration on a C18 solid-phase extraction column and reaction with benzoic anhydride at 50°C and with 1,2,4-triazole and mercury(II) chloride solution, pH 9.0, at 65°C. The derivatized compounds are eluted isocratically on a C8 column with a mobile phase of acetonitrile and phosphate buffer (pH 6; 0.1M) containing sodium thiosulfate and the ion-pair reagent tetrabutylammonium hydrogen sulfate. The penicillins are detected by UV absorption at 325 nm. The limit of detection and the limit of determination (quantitation) of the method were calculated to be approximately 3–5 and 25 μg/kg, respectively, in accordance with the criteria of European Union (EU) Decision No. 93/256/EEC. In this first interlaboratory study, collaborators were instructed to monitor 4 different penicillin compounds (benzylpenicillin, phenoxymethylpenicillin, ampicillin, and amoxicillin) by analyzing 8 blind samples of muscle tissue in triplicate. These samples were prepared from 2 materials containing different concentrations of incurred ampicillin (63.5 μg/kg for material No. 1 and 358.1 μg/kg for material No. 2) and 1 blank material. The repeatability relative standard deviation and the reproducibility relative standard deviation were 10.2 and 17.4%, respectively, for material No. 1 and 7.0 and 16.0%, respectively, for material No. 2. These results demonstrate that the method is suitable for the determination of ampicillin residues in muscle tissue at the EU maximum residue limit (50 μg/kg) and above. However, the identification of positives by this procedure may need additional confirmation by techniques with greater specificity, such as liquid chromatography combined with mass spectrometry, or tandem mass spectrometry. Investigations regarding the basis of interlaboratory testing studies will further demonstrate the suitability of multiresidue methodology for detecting and quantitating other compounds in the family of penicillin antibiotics.

A biosensor-based immunoassay for the screening of penicillin residues in milk was developed. The BIAcore biosensor which was employed was based on surface plasmon resonance detection. One commercial antibody against ampicillin which had much higher affinity for open beta-lactam ring than for closed ring was chosen. Two different pre-treatments were tested prior to the biosensor assay to open the ring in order to increase the assay sensitivity. These two ways were enzymatic (penicillinase) and chemical pre-treatment of the samples. Limits of detection for ampicillin in milk were 33 and 12.5 μg l −1 after enzymatic and chemical pre-treatment, respectively. There were no cross-reactions with cephalosporins and other families of antibiotics or proteins in the buffer and in milk after both the pre-treatments. Percentages of cross-reactivity with nine tested penicillins were often clearly higher in milk than in buffer. Moreover, a lack of sensitivity after penicillinase pre-treatment was observed. Conversely, penicillins G and penicillins M could be detected at or below their respective MRLs (4 and 30 μg l −1 in milk) and ampicillin and amoxicillin could be detected only at about three MRLs after chemical pre-treatment. However, the enzymatic pre-treatment was much easier to perform and led to more stable results.

Biosensor immunoassays were developed recently for antibiotics with an established maximum residue limit (MRL). In this study, according to the regulatory banning of chloramphenicol (CAP) use for food producing animals, the main objectives were: the specificity of the biosensor assay and the lowest detection limit possible. The assay was based on the inhibition of the binding of polyclonal antibodies against CAP to immobilized CAP on a sensor chip by CAP in solution. The response varied inversely with the antibiotic concentration in the sample. Two different antibodies and two immobilization protocols were tested. As in ELISA tests the antibody influenced the assay performances. Moreover, we showed that particular care should be concentrated on the immobilization step because it is a critical point in the assay development. Three different protocols were developed in milk. The best assay was obtained with antibody 1 in milk on the CAP base surface because of its very low detection limit (0.1 w g l−1) and ...

Aims The objective of this study was to evaluate fungicidal efficacy of hydrogen peroxide administered in combination with 17 mineral and organic acids authorized for use in the food industry. Methods and Results The assays were performed on a 96-well microplate using a microdilution technique based on the checkerboard titration method. The six selected strains (one yeast and five fungi) were reference strains and strains representative of contaminating fungi found in the food industry. Each synergistic hydrogen peroxide/acid combination found after fifteen minutes contact time at 20°C in distilled water was then tested in conditions simulating four different use conditions. Twelve combinations were synergistic in distilled water, eleven of these remained synergistic with one or more of the four mineral and organic interfering substances selected. Hydrogen peroxide/formic acid combination remained effective against four strains and was never antagonistic against the other two fungi. Combinations with propionic acid and acetic acid stayed synergistic against two strains. Those with oxalic acid and lactic acid kept their synergism only against Candida albicans. No synergism was detected against Penicillium cyclopium. Conclusions Synergistic combinations of disinfectants were revealed, among them the promising hydrogen peroxide/formic acid combination. Significance and Impact of the Study A rapid screening method developed in our laboratory for bacteria was adapted to fungi and used to reveal the synergistic potential of disinfectants and/or sanitizers combinations.

Aims The objective of this study was to evaluate the bactericidal efficacy of hydrogen peroxide administered in combination with 17 mineral and organic acids authorized for use in the food industry. Methods and Results The assays were performed on a 96-well microplate using a microdilution technique based on the checkerboard titration method. The six selected strains were reference strains and strains representative of contaminating bacteria in the food industry. Each synergistic hydrogen peroxide/acid combination found after 5-min contact time at 20°C in distilled water was then tested in conditions simulating four different use conditions. Thirty-two combinations were synergistic in distilled water; twenty-five of these remained synergistic with one or more of the four mineral and organic interfering substances selected. Hydrogen peroxide/formic acid combination was synergistic for all six bacterial strains in distilled water and remained synergistic with interfering substances. Six other combinations maintained their synergistic effect in the presence of an organic load but only for one or two bacterial strains. Conclusions Synergistic combinations of disinfectants were revealed, among them the promising hydrogen peroxide/formic acid combination. Significance and Impact of the Study A rapid screening method was proposed and used to reveal the synergistic potential of disinfectant and/or sanitizer combinations.

Bacterial adaptation to quaternary ammonium compounds (QACs) is mainly documented for benzalkonium chloride (BC) and few data are available for other QACs. The aim of this study was to assess the effects of repeated exposure to different quaternary ammonium compounds (QACs) on the susceptibility and/or resistance of bacteria to other QACs and antibiotics. Escherichia coli strains (n=10) were adapted by daily exposure to increasingly sub-inhibitory concentrations of a QAC for 7 days. Three QACs were studied. Following adaptation, we found similar levels of reduction in susceptibility to QACs with a mean 3-fold increase in the minimum inhibitory concentration (MIC) compared to initial MIC values, whatever the QAC used during adaptation. No significant differences in antibiotic susceptibility were observed between the tested QACs. Antibiotic susceptibility was reduced from 3.5- to 7.5-fold for phenicol compounds, β lactams, and quinolones. Increased MIC was associated with a shift in phenotype from susceptible to resistant for phenicol compounds (florfenicol and chloramphenicol) in 90% of E. coli strains. Regardless of the QAC used for adaptation, exposure to gradually increasing concentrations of this type of disinfectant results in reduced susceptibility to QACs and antibiotics as well as cross-resistance to phenicol compounds in E. coli strains. Extensive use of QACs at sub-inhibitory concentrations may lead to the emergence of antibiotic-resistant bacteria and may represent a public health risk.

Campylobacter is responsible for human bacterial enteritis and poultry meat is recognised as a primary source of infection. In slaughterhouses, cleaning and disinfection procedures are performed daily, and it has been suggested that disinfectant molecules might select for antibiotic resistant strains if shared targets or combined resistance mechanisms were involved. The aim of the study was to investigate if cleaning and disinfection procedures in poultry slaughterhouses select for antibiotic resistance in Campylobacter jejuni and C. coli and to determine the genotypes of isolates collected after cleaning and disinfection. Nine sampling visits were made to four French slaughterhouses. Samples were collected from transport crates and equipment surfaces, before and after cleaning and disinfection. Minimal inhibitory concentrations of the recovered C. jejuni and C. coli isolates to six antibiotics and two disinfectants were measured. The C. jejuni isolates collected from equipment surfaces after cleaning and disinfection were subjected to PCR-RFLP typing. Twenty-five C. jejuni isolates and 1 C. coli were recovered from equipment surfaces after cleaning and disinfection during five visits to three different slaughterhouses. Those isolates did not show an increased resistance to the tested antibiotics compared to isolates collected before cleaning and disinfection. Only one or two genotypes were recovered after cleaning and disinfection during single visits to each slaughterhouse. This observation suggests that such genotypes may be particularly adapted to survive cleaning and disinfection stress. Understanding the survival mechanisms of Campylobacter should facilitate the implementation of better-targeted strategies and reduce the public health burden associated with Campylobacter infection.

A number of commercially available disinfectants are commonly used on pig breeding farms and are authorised by the French Agricultural Ministry. However, the efficacy of these disinfectants is unknown with regard to the emergent porcine circovirus type 2 (PCV2). The virucidal efficacy of nine disinfectants was evaluated by testing a suspension of PCV2 isolated in France. The assays were performed at 20 degrees C and the efficacy determined after 30 min contact time between virus and disinfectant. After this time, the mixture was passed through a detoxification column and then diluted to remove compounds toxic to the virus and the porcine kidney cell line. The filtrate was serially diluted and inoculated onto cell culture. The infectivity of PCV2 was determined by an immunoperoxidase monolayer assay. No reduction in PCV2 titre was demonstrated with iodine and phenolic products. Significant PCV2 titre reductions (1.61 log(10)) were noted for the seven other products. For five disinfectants, namely a product composed of potassium monopersulfate, two products comprising a quaternary ammonium with one or three aldehyde(s), sodium hypochlorite, and sodium hydroxide, the concentration that significantly reduced the PCV2 titre was equal or 1.5-4 times lower than the authorised use concentration. Only two disinfectants, one composed of potassium monopersulfate, the other containing peracetic acid with hydrogen peroxide, reduced the PCV2 titre with a product concentration at best equal or two times higher than the authorised use concentration.

Maris Pierre. Les désinfectants. Efficacité et niveaux d’exigences. In: Bulletin de l'Académie Vétérinaire de France tome 155 n°2, 2002. pp. 19-28.