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This study was conducted to evaluate the performance of a screening protocol to detect and isolate mcr-positive Escherichia coli and Salmonella spp. from animal caecal content and meat samples. We used a multicentre approach involving 12 laboratories from nine European countries. All participants applied the same methodology combining a multiplex PCR performed on DNA extracted from a pre-enrichment step, followed by a selective culture step on three commercially available chromogenic agar plates. The test panel was composed of two negative samples and four samples artificially contaminated with E. coli and Salmonella spp. respectively harbouring mcr-1 or mcr-3 and mcr-4 or mcr-5 genes. PCR screening resulted in a specificity of 100% and a sensitivity of 83%. Sensitivity of each agar medium to detect mcr-positive colistin-resistant E. coli or Salmonella spp. strains was 86% for CHROMID(R)Colistin R, 75% for CHROMagarTM COL-APSE and 70% for COLISTIGRAM. This combined method was effective to detect and isolate most of the E. coli or Salmonella spp. strains harbouring different mcr genes from food-producing animals and food products and might thus be used as a harmonized protocol for the screening of mcr genes in food-producing animals and food products in Europe.

Microbial spoilage of liquid egg products and its control by the detection of genetic or chemical markers. journée scientifique "science des aliments et valorisation des bio-produits"

Conference: 11th International Symposium on Biometals (Biometals) Location: Ottawa, CANADA Date: 2018; Salmonella enterica serovar Enteritidis (SE) is the most frequently-detected Salmonella in foodborne outbreaks in the European Union. Among such outbreaks, egg and egg products were identifiedas the most common vehicles of infection. Possibly, the major antibacterial property of egg white is ironrestriction, which results from the presence of the ironbinding protein, ovotransferrin. To circumvent ironrestriction, SE synthesise catecholate siderophores (i.e. enterobactin and salmochelin) that can chelateiron from host iron-binding proteins. Here, we highlight the role of lipocalin-like proteins found in eggwhite that could enhance egg-white iron restriction through sequestration of certain siderophores, includingenterobactin. Indeed, it is now apparent that the egg-white lipocalin, Ex-FABP, can inhibit bacterialgrowth via its siderophore-binding capacity in vitro. However, it remains unclear whether Ex-FABP performssuch a function in egg white or during bird infection. Regarding the two other lipocalins of eggwhite (Cal-c and a-1-glycoprotein), there is currently no evidence to indicate that they sequestersiderophores.

National audience; Salmonella outbreaks are a serious threat to public health. One of the main vehicles of infection are eggs and egg-products, with Salmonella enterica serovar Enteritidis being the most frequent contaminant. Egg contamination occurs despite the impressive antibacterial properties of egg white and it is notable that S. Enteritidis is able to survive in egg white in contrast to other serovars and species. It is generally accepted that the major egg white-factor limiting growth of Salmonella is the highly iron-restricted environment resulting from the presence of ovotransferrin, an iron-binding protein. To circumvent iron restriction, many bacteria synthesise siderophores that can chelate iron from host iron-binding proteins for delivery to bacteria. The iron starvation response genes are regulated according to iron availability via the transcriptional regulator, Fur. Evidence for a relationship between temperature and iron sequestration ability has been reported by several groups. For Salmonella Typhymurium, little or no siderophore is secreted at 40-42°C in iron depleted medium indicating a temperature regulatory effect that is independent of Fur/Fe control. The aim of this study is to determine if egg white exposure induces the iron-acquisition genes and siderophore production in the highly-egg-white adapted Enteritidis serovar at ~40°C, the natural hen body temperature that Salmonella would encounter when infecting the egg during egg formation in the oviduct or during hatching. In order to easily recover of S. Enteritidis, we used egg white model medium (EWMM) in place of viscous egg white. EWMM consists of egg white filtrate with 10% egg white and numerous studies have validated this model medium. We measured the expression of 15 genes involved in the ironrestriction response by qRT-PCR after S. Enteritidis incubation of 45 minutes in EWMM at 37, 40 and 42°C. We observed a strong expression of genes encoding proteins involved in biosynthesis, export, uptake and utilisation of enterobactin and salmochelin (the two siderophores of Salmonella) and ferric-hydroxamate uptake. A statistical analysis shows that iron-acquisition genes are better expressed at 40°C than at 37 or 42°C. The production of siderophores was quantified during incubation using the Calcein blue test. Siderophores released by Salmonella in EWMM were detected at around a 1 μM desferral equivalent within 6h of incubation at 37, 40 and 42°C. The results of this study suggest that S. Enteritidis is able to induce its iron acquisition systems to cope with the iron-restricted conditions imposed by egg white even at the elevated body temperature of the hen.

Les anevrismes infectieux de l'axe femoro-poplite sont rares et d'evolution silencieuse. Ils surviennent surtout chez les sujets immunodeprimes. Leur traitement fait appel souvent a l'exclusion chirurgicale associee ou non a un geste de revascularisation. Nous rapportons le cas d'un patient opere pour un anevrisme de l'artere femorale superficielle, lie a une infection a salmonelle, il a eu une resection de l'anevrysme et une revascularisation par greffe veineuse. Ce patient a ete repris par la suite a deux reprise pour rupture du greffon veineux. Pour cette raison, nous etions contraints a une explantation du pontage et une ligature des 2 bouts de l'artere sans revascularisation. Malgre l'absence de revascularisation, l'evolution a ete favorable sous couverture d'une une antibiotherapie adaptee. En cas d'anevrysme infectieux de l'axe femoropoplite par infection a salmonelle, le retablissement de la continuite arterielle, meme avec un greffon veineux, peut exposer au risque de rupture arterielle. Key words: Anevrisme mycotique, artere femorale superficielle, salmonelle

As a 1st step, this study aimed at investigating the microbial quality of liquid egg white in a French eggprocessing company. Thirty raw and 33 pasteurized liquid egg white samples were analyzed. Pasteurization was globally found efficient on mesophilic contaminants (1.7 ± 1.6 and 0.8 ± 0.9 log CFU/mL in raw and pasteurized samples, respectively), including for the control of Salmonella. However,Gram-positive enterococci were still detected in the pasteurized samples. As a 2nd step, a representative bacterial collection was built for exploring the spoilage issue in egg-based chilled desserts. Custard cream was chosen as growth medium since this food is widely used for the production of French chilled desserts. All of the 166 isolates of the bacterial collection were shown to be able to grow and to induce spoilage of the custard cream at refrigeration temperature (10 °C). Several spoilage types were highlighted in the custard cream, on the basis of changes regarding pH, consistency, production of holes or gas. As a 3rd step, bacterial enzymatic activities were explored on custard cream-based agar media. The bacterial collection was reduced to 43 isolates, based on further selection regarding the genera and the spoilage types previously highlighted. Albeit to different degrees, all these isolates were able to produce proteases. A large part of these isolates also expressed lipolytic and amylolytic activities. This studyemphasizes the need to control egg white contamination and especially with Gram-positive heat-resistant Enterococi, in order to guarantee the shelf life of egg-based chilled desserts.

Liste des auteurs cf: EFSA Panel on Biological Hazards (BIOHAZ): biohaz@efsa.europa.euEuropean Food Safety Authority (EFSA); Salmonella Enteritidis is considered the only pathogen currently posing a major risk of egg-borne diseases in the European Union (EU). The possible impact of extending the shelf-life of eggs on the risk to consumers posed by S. Enteritidis was estimated by applying a quantitative model and comparing the actual situation regarding the storage of eggs in the EU with different possible scenarios combined, considering the prolongation of the best-before and the sell-by date from 7 to a maximum of 70 days. Extending the sell-by date by one week (from 21 to 28 days), but leaving the best-before date unchanged, is estimated to result in a relative risk of illness of 1.4 and 1.5 for uncooked and lightly cooked egg meals respectively, compared to the current situation. If the best-before date is also extended by one week (from 28 to 35 days), the relative risk would be 1.6 and 1.7. In the worst case scenario considered (sell-by date of 42 days, best before date of 70 days), such figures would be 2.9 and 3.5. It should be noted that the absolute risk is greater for uncooked meals compared to lightly cooked meals. An effective way to minimise any increase in risk during extended storage is to keep the eggs refrigerated both at retail and the household. Regarding egg spoilage, such events strongly depend on the hygienic conditions of egg production and practices of egg handling, including storage times and temperatures. Finally, the impact of the prolongation of storage time on the quality criteria for eggs (3-hydroxybutyric acid and lactic acid) destined for manufacturing of egg products is considered negligible.

All over the world, the incidence of Salmonella spp contamination on different food sources like broilers, clams and cow milk has increased rapidly in recent years. The multifaceted properties of Salomnella serovars allow the microorganism to grow and multiply in various food matrices, even under adverse conditions. Therefore, methods are needed to detect and trace this pathogen along the entire food supply network. In the present work, PFGE and ERIC-PCR were used to subtype 45 Salmonella isolates belonging to different serovars and derived from different food origins. Among these isolates, S. Enteritidis and S. Kentucky were found to be the most predominant serovars. The Discrimination Index obtained by ERIC-PCR (0.85) was slightly below the acceptable confidence value. The best discriminatory ability was observed when PFGE typing method was used alone (DI = 0.94) or combined with ERIC-PCR (DI = 0.93). A wide variety of profiles was observed between the different serovars using PFGE or/and ERIC-PCR. This diversity is particularly important when the sample origins are varied and even within the same sampling origin.

International audience

Increasing resistance to acute Salmonellosis (that is, contamination level shortly after infection) is not sufficient to reduce the risk for consumers to be contaminated by Salmonella. Indeed, animals may remain contaminated at a low level for weeks or months. Increased resistance to the Salmonella carrier state, i.e., animals' ability to clear bacteria, is needed; it involves measuring bacterial contamination several weeks after inoculation with a low dose. To study such resistance traits, three convergent approaches were used. A quantitative trait loci (QTL) study was performed, taking advantage of inbred lines differing in resistance. Several QTLs controlling resistance at a younger age were identified and are currently being confirmed in a new cross before finer mapping, using advanced intercross lines. These inbred lines are also presently being compared using functional genomics. In parallel, a selection experiment for increased or decreased resistance at a younger and a later age was undertaken. Besides providing genetic models differing in their levels of resistance, it underlined the importance of the choice of selection criterion, whether marker assisted or not. Indeed, genes controlling resistance are strongly dependant on age; selecting for resistance at a younger age might result in increased susceptibility at an older age. Finally, the results of this experiment were used in a model of the intra-flock propagation of Salmonella. It showed that introducing a proportion of resistant animals within a flock of susceptible hens could dramatically change the evolution of contamination. Moreover, it demonstrated the magnitude of synergy between selection and vaccination, which should enhance the interest of increased resistance. The results show that selection for increased resistance to the Salmonella carrier state may be efficient, providing that the appropriate criteria of selection are used.

International audience; Reliable ELISAs were investigated with the aim to select hen lines resistant to Salmonella Enteritidis and producing high levels of antibodies. In the first experiment, the relation between the humoral response and the bacteriological results was assessed on hens from the Y11 resistant line and the L2 susceptible line, orally inoculated with 10$^8$ CFU S. Enteritidis per animal. Anti- lipopolysaccharide (LPS) IgG titres were higher but the liver and spleen were less contaminated in hens from the Y11 line than in hens from the L2 line ($p = 0.013$, 0.031 and 0.026 respectively). In the second experiment, the hens were inoculated orally with $1.7 \times 10^8$ CFU S. Enteritidis per animal in order to select the ELISA methods showing the more significant differences. ELISAs were based on LPS, flagella, LPS from rough (LPS-R) and smooth strains (LPS-S) and detected IgG and IgM antibodies from sera and yolks. No between-line host response variation was observed in the yolk, with LPS-S and R antigens nor with anti-LPS IgM in the sera. Otherwise, significant differences were encountered between hen lines with the ELISAs performed on the sera detecting anti-LPS IgG, anti-flagella IgG or IgM ($p = 0.017$, 0.017 and $p < 0.001$ respectively). When comparing the kinetics of the selected ELISAs, the IgG antibodies against LPS detected between-line variations as early as 1 to 4 weeks pi, whereas with IgG against flagella, the differences were only detected at 1 and 2 weeks pi and with IgM against flagella, the differences were significant at 1, 2, 4 and 8 weeks pi. In conclusion, resistant hen lines producing higher levels of antibodies than the susceptible hen lines may be selected with these ELISAs.; Méthodes ELISA permettant de différencier des lignées de poules résistante et sensible à Salmonella Enteritidis inoculées par voie orale. Des méthodes ELISA ont été recherchées pour sélectionner les lignées de poules résistantes à Salmonella Enteritidis produisant des taux élevés d'anticorps. Dans la première expérience, la relation entre la réponse sérologique et les résultats bactériologiques a été évaluée sur les poules des lignées résistante Y11 et sensible L2, inoculées par voie orale avec 10$^8$ UFC S. Enteritidis par animal. Les titres en anticorps IgG anti- lipopolysaccharidiques (LPS) étaient supérieurs mais les foies et rates étaient moins contaminés chez les poules de la lignée Y11 que chez les poules de la lignée L2 ($p = 0.013$, 0.031 et 0.026 respectivement). Dans la seconde expérience, les deux lignées de poules ont été inoculées par voie orale avec $1.7 \times 10^8$ UFC S. Enteritidis par animal afin de sélectionner les méthodes ELISA les plus discriminantes. Les ELISA utilisaient comme antigènes des LPS, flagelles, LPS des souches rough (LPS-R) et smooth (LPS-S), et détectaient des anticorps IgG et IgM dans le sérum et le vitellus. Aucune différence significative n'a été observée à partir du vitellus, ou avec les antigènes LPS-S et R, ainsi qu'avec les IgM anti-LPS dans le sérum. En revanche, les tests ELISA réalisés sur les sérums et basés sur le dépistage des IgG anti-LPS, des IgG ou IgM anti-flagellaires permettent de montrer une différence significative entre les deux lignées ($p = 0.017$, 0.017 et $p < 0.001$ respectivement). La comparaison des cinétiques obtenues avec les tests ELISAs retenus a montré que les IgG anti-LPS permettaient de détecter des différences entre lignées dès 1 semaine jusqu'à 4 semaines après inoculation, alors qu'avec les IgG anti-flagellaires les différences apparaissaient seulement à 1 et 2 semaines, et avec les IgM anti-flagellaires les différences étaient significatives à 1, 2, 4 et 8 semaines après inoculation. En conclusion, les lignées de poules résistantes et produisant des taux d'anticorps plus élevés que les lignées de poules sensibles peuvent être sélectionnées avec ces tests ELISA.

International audience

Chantier qualité GA; International audience; UNLABELLED: ABSTRACT: BACKGROUND: Chicken meat and eggs can be a source of human zoonotic pathogens, especially Salmonella species. These food items contain a potential hazard for humans. Chickens lines differ in susceptibility for Salmonella and can harbor Salmonella pathogens without showing clinical signs of illness. Many investigations including genomic studies have examined the mechanisms how chickens react to infection. Apart from the innate immune response, many physiological mechanisms and pathways are reported to be involved in the chicken host response to Salmonella infection. The objective of this study was to perform a meta-analysis of diverse experiments to identify general and host specific mechanisms to the Salmonella challenge. RESULTS: Diverse chicken lines differing in susceptibility to Salmonella infection were challenged with different Salmonella serovars at several time points. Various tissues were sampled at different time points post-infection, and resulting host transcriptional differences investigated using different microarray platforms. The meta-analysis was performed with the R-package metaMA to create lists of differentially regulated genes. These gene lists showed many similarities for different chicken breeds and tissues, and also for different Salmonella serovars measured at different times post infection. Functional biological analysis of these differentially expressed gene lists revealed several common mechanisms for the chicken host response to Salmonella infection. The meta-analysis-specific genes (i.e. genes found differentially expressed only in the meta-analysis) confirmed and expanded the biological functional mechanisms. CONCLUSIONS: The meta-analysis combination of heterogeneous expression profiling data provided useful insights into the common metabolic pathways and functions of different chicken lines infected with different Salmonella serovars.