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See detailStudy of the microbial flora of freshwater and seawater fish filets in different packaging conditions by metagenomic analysis targeted on the 16S ribosomal DNA
Delhalle, Laurent ULg; Taminiau, Bernard ULg; Nezer, Carine et al

Conference (2012, October 19)

Metagenomics has appeared as a powerful tool to study bacterial composition of various environmental samples. This work describes the application of this technique to study the bacterial population of two ... [more ▼]

Metagenomics has appeared as a powerful tool to study bacterial composition of various environmental samples. This work describes the application of this technique to study the bacterial population of two fresh fish filets. The two fish species are from freshwater (pangasius) and seawater (haddock), respectively. Samples where directly analyzed the day of receipt. Others samples were analyzed at the end their shelf life after storage at 4°C (1/3 of their shelf life) and 8°C (2/3 of their shelf life). For these samples, packagings were made in plastic wrap for atmospheric air condition and in trays under modified atmosphere. Classical microbiological and 16S rDNA metagenomic analysis were carried out on all these samples. The composition and evolution of microbial populations of fish filet stored under different packaging conditions and temperatures of storage were investigated with identification of bacteria species. A total of 40 different species were identified for both fish types. Gram-negative bacteria are always predominated among the initial flora and at the end of the shelf life in all the trials. At the beginning of storage, the predominant Gram-negative microflora consisted of Moraxellaceae (Acinetobacter spp, Psychrobacter spp.), Pseudomonadaceae (Pseudomonas spp), and Shewanella spp and the Gram-positive flora was identified as Lactobacillaceae (Carnobacterium spp), Brochothrix thermosphacta and Planococcus donghaensis (only for pangasius). For the pangasius, Planococcus donghaensis is only present before the fish is packed and its dominant presence could provide an indication of the freshness of the fish. The metagenomic analysis is a useful tool to identify and to measure the relative proportions of bacterial species in fish filet samples. [less ▲]

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See detailEffect of duration and temperature of previous vacuum-packed storage on the microbiological quality of Belgian Blue meat packed in high-oxygen atmosphere
Didimo Imazaki, Pedro Henrique ULg; Tahiri, Assia ULg; Daube, Georges ULg et al

Poster (2012, September 05)

The aim of this experiment was to study the effect of duration and temperature of previous vacuum-packed (VP) storage on the microbiological quality of Belgian Blue (BB) beef packed in high-oxygen ... [more ▼]

The aim of this experiment was to study the effect of duration and temperature of previous vacuum-packed (VP) storage on the microbiological quality of Belgian Blue (BB) beef packed in high-oxygen atmosphere. VP striploins from bulls (B) and cows (C) were stored at −1 °C and +4 °C for up to 80 days. These meats were subsequently repackaged under modified atmosphere (MA) – 70 % O2/30 % CO2 – at different times, and stored 2 d at +4 °C and 5 d at +8 °C. The average initial counts in VP meats were 3.6 log CFU/cm² (B) and 2.7 log CFU/cm² (C) for total viable count (TVC) at +22 °C; < 2.0 log CFU/cm² (B and C) for lactic ac id bacteria (LAB) at +22 °C; 1.1 log CFU/cm² (B) and 1.3 log CFU/cm² (C) for Enterobacteriaceae at +30 °C and < 1.0 log CFU/cm² (B and C) for Pseudomonas spp. and Brochothrix thermosphacta. During the first 40 days of VP storage, temperature had a striking influence on microbial growth. The maximum count differences between storage temperatures were obtained at the 20th day of storage: 2.7 log CFU/cm² (B) and 2.9 log CFU/cm² (C) for TVC, 4.0 log CFU/cm² (B and C) for LAB and 3.6 log CFU/cm² (B and C) for Enterobacteriaceae. The difference in TVC between temperatures at the 20th day tended to disappear once the meats were repacked under MA and stored during seven days. Conversely, the difference in LAB and Enterobacteriaceae counts tended to be maintained after MA repackaging, showing that duration and temperature of VP storage had influence on microbiological quality of BB meat subsequently stored in high-oxygen atmosphere. Moreover, chilling at temperatures very close to the freezing point of meat during VP storage, which has already showed innumerous advantages for physicochemical quality of meat, was capital to maintain the microbiological quality of BB fresh meat during subsequent MA-packed storage. [less ▲]

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See detailMetagenomic analysis as a tool to better characterize the bacterial content of food and food preparations.
Taminiau, Bernard ULg; Delhalle, Laurent ULg; Nezer, Carine et al

Conference (2012, September 04)

Metagenomic analysis is a new culture-independent approach for assigning a taxonomic, genic or functional identity to bacterial DNA fragments of unknown origin. Its power and utility is increasingly ... [more ▼]

Metagenomic analysis is a new culture-independent approach for assigning a taxonomic, genic or functional identity to bacterial DNA fragments of unknown origin. Its power and utility is increasingly rising thanks to the next generation sequencing techniques. It is now mature and cheap enough to be transposed to more applied fields like the food microbiology. We demonstrated in several studies the extraordinary potential of the targeted metagenomic analysis to different problematics related to food products. First, this approach is highly useful for the validation of the shelf life of food products. We analyzed standardized pork minced meat and meat product samples packaged either under modified atmosphere (MAP - 30% CO2, 70% O2) or under permeable atmosphere packaging, stored at different temperature (4°C, 4-8°C and 12°C) until the end of shelf-life. The metagenomic analysis allowed to identify species of all the sub-dominant bacterial populations. This approach showed why MAP can improve meat quality by favoring certain species rather than others. As a second example, we sought to identify the potential spoiling bacteria in several food products like raw fish, rind cheese or vacuum packed beef meats in order to illustrate the usefulness of metagenomics for the quality control of food preparations. Samples from various food matrices were screened to identify the bacterial contaminants. We combined the bioinformatics analysis with a classical approach to generate effective quantitative data for the various bacterial populations detected. This analysis characterizes the samples both on the identity of the potential spoiling bacteria present and on the quantification level of the contaminants. Finally, the metagenomic analysis reveals the presence of numerous uncultured and uncharacterized bacteria. The use of a carefully designed analysis pipeline has been used to ensure to label the bacterial population with a precise taxonomic identity and to determine whether the targeted population corresponds to a known species or not. This way, even if the nearest known homologous sequence is an environmental sample, its relatedness to known species can be deduced. This represents a new tool to trace yet uncharacterized food spoiling bacteria. [less ▲]

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See detailMolecular identification of the bacterial populations of steak tartare, a raw consumed meat preparation: a practical use of targeted metagenomic analysis
Taminiau, Bernard ULg; Delhalle, Laurent ULg; Nezer, Carine et al

Poster (2012, September 04)

Steak tartare is a popular meat dish in Belgium and other european countries. It is often consumed with french fries or as sandwich spread. This product, due to its raw nature, is highly sensitive to ... [more ▼]

Steak tartare is a popular meat dish in Belgium and other european countries. It is often consumed with french fries or as sandwich spread. This product, due to its raw nature, is highly sensitive to bacterial alteration. A better understanding of the bacterial content of this meat product will thus be insightful to master the alteration hazards. Throughout a targeted metagenomic analysis we characterized the bacterial populations of several steak tartare samples. These samples were bought and analyzed during the same day, from three different commercial sources: butchery, sandwich vendor and restaurant. A classic microbiological analysis was performed in parallel. The metagenomic analysis was targeted on two different hypervariable regions of the bacterial 16S rDNA, in order to compare the bacterial identification efficiency. A total of 60,500 sequences for 12 samples were submitted to a metagenomic analysis. The best hypervariable region enabled us to identify 356 different bacterial species. Lactobacillus algidus is the leading bacterial species, representing 52% of the total analyzed sequences, followed by an uncultured Pseudomonas sp. (8.43%) and Photobacterium phosphoreum (7.92%). The analysis of the results shows that remarkable differences appear between the three sources of steak tartare. First, the samples from the butchery are mainly composed of Lactobacillus populations and to a lesser extend of environmental contaminants like Xanthomonas campestris. On the opposite, the samples from the restaurant are contaminated with higher level of Leuconostocaceae like Leuconostoc carnosum or an uncultured Weissella sp., or with gamma-proteobacteria like Pseudomonas sp. or Psychrobacter sp. These last samples were characterized with some alteration (slime, off odor) that can thus be put in relation with the bacterial populations identified. Combining a broad-range sequencing effort to a rigorous computer analysis gives a powerful tool for the microbiology of food products. Its application can be virtually extended to every food product be readily transposed to the food industry. [less ▲]

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See detailThe extraordinary potential of metagenomic tools for food microbiology: an example with bacterial microbiota of raw and pasteurized milk cheeses
Delhalle, Laurent ULg; Nezer, Carine; Taminiau, Bernard ULg et al

Poster (2012, September 03)

Among the culture-independent techniques, ultra-sequencing has contributed to place metagenomic analysis as the best alternative to study complex microbiota. During the last three years, metagenomic ... [more ▼]

Among the culture-independent techniques, ultra-sequencing has contributed to place metagenomic analysis as the best alternative to study complex microbiota. During the last three years, metagenomic studies were used essentially for environmental samples but it could be used also to analyse bacterial populations of food samples. This work describes the application of this technique to study the bacterial population of different types of soft cheeses. Among these, three of them are a typical Belgian soft cheese with washed rind (two with raw milk and the third with pasteurized milk). The fourth is a French creamy soft cheese made with raw milk. Classical microbiological and 16S rDNA metagenomic analysis were carried out in the core and on the rind of the four cheeses, giving a total of 8 samples. In total, 48 genus and 163 species were identified for all samples. As expected Lactoccocus lactis and/or cremoris are the most representative species in the core of the four cheeses. On the rind of cheeses, the predominant bacterial species are Psychrobacter glacinola, Staphylococcus equorum, Corynebacterium casei and Marinilactibacillus psychrotolerans. Brevibacterium spp and Psychroflexus spp are important for the rind of washed rind cheeses. All these species are present in different proportions following the origin and the cheese making process and they are well known for their organoleptic properties on the rind of cheese. The two Belgian soft cheese made with raw milk are composed of many more different bacterial species. While the cheese made from pasteurized milk contains less species mainly composed by Lactococcus lactis (97,6%) in the core. An unexpected result is the low diversity of the a French creamy soft cheese made with raw milk with only two predominent species : Lactoccocus cremoris and Leuconostoc citreum are present in the core (94,9% and 4,9% , respectively) and on the rind (93,8% and 5% , respectively). Compared with the other cheeses made with raw milk, this result is surprising. The bacterial cheese microbiota plays a central role in cheese-making. The subtleties of cheese character, as well as their shelf-life, are largely determined by the evolution of their microbiota. [less ▲]

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See detailA new approach of food microbiology with the metagenomic tools: an application on fish
Delhalle, Laurent ULg; Taminiau, Bernard ULg; Nezer, Carine et al

Poster (2012, September 03)

Metagenomics has appeared as a powerful tool to study bacterial composition of various environmental samples. Its interest has started to appear in food microbiology but only on the study of very ... [more ▼]

Metagenomics has appeared as a powerful tool to study bacterial composition of various environmental samples. Its interest has started to appear in food microbiology but only on the study of very particular bacterial populations of fermented food. This work describes the application of this technique to study the bacterial population of two fresh fish filets. The two fish species are from freshwater (pangasius) and seawater (haddock), respectively. Samples where directly analyzed the day of receipt. Others samples were analyzed at the end their shelf life after storage at 4°C (1/3 of their shelf life) and 8°C (2/3 of their shelf life). For these samples, packagings were made in plastic wrap for atmospheric air condition and in trays under modified atmosphere. Classical microbiological and 16S rDNA metagenomic analysis were carried out on all these samples. The composition and evolution of microbial populations of fish filet stored under different packaging conditions and temperatures of storage were investigated with identification of bacteria species. A total of 40 different species were identified for both fish types. Gram-negative bacteria are always predominated among the initial flora and at the end of the shelf life in all the trials. At the beginning of storage, the predominant Gram-negative microflora consisted of Moraxellaceae (Acinetobacter spp, Psychrobacter spp.), Pseudomonadaceae (Pseudomonas spp), and Shewanella spp and the Gram-positive flora was identified as Lactobacillaceae (Carnobacterium spp), Brochothrix thermosphacta and Planococcus donghaensis (only for pangasius). Regardless the packaging and the fish origin, significant variations of the initial flora were noted. The important growth of some Gram negative populations could indicate a risk of spoilage. Thus, the metagenomic approach could be used to adequately determine the duration of shelf-life. For the pangasius, Planococcus donghaensis is only present before the fish is packed and its dominant presence could provide an indication of the freshness of the fish. The metagenomic analysis is a useful tool to identify and to measure the relative proportions of bacterial species in fish filet samples. [less ▲]

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See detailLes modèles de microbiologie prévisionellepour la maitrise de la sécurité des aliments (synthèse bibliographique)
Delhalle, Laurent ULg; Daube, Georges ULg; Adolphe, Ysabelle ULg et al

in Biotechnologie, Agronomie, Société et Environnement = Biotechnology, Agronomy, Society and Environment [=BASE] (2012), 16(3), 369-381

Predictive microbiology aims to predict the evolution of microorganisms in foods with mathematical models. Several models have been published and the complexity of some of them makes their use difficult ... [more ▼]

Predictive microbiology aims to predict the evolution of microorganisms in foods with mathematical models. Several models have been published and the complexity of some of them makes their use difficult for the uninitiated. However, the use of this discipline will become widespread in coming years. These models provide, for example, additional tools to ensure the microbiological safety of food, to establish the contamination flow in a food chain, to develop and to assist the quality assurance systems. The development of new computer software and database will enable stakeholders in the food chain to have a better control of microbiological hazards. The aim of this summary is to give an overview of existing models of predictive microbiology and their applications. A first approach of the primary, secondary and tertiary models is given. The modelling of latency, integrated models and growth tests are also discussed. [less ▲]

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See detailThe Metagenomic in the Service of the Food Microbiology.
Taminiau, Bernard ULg; Nezer, Carine; Poullet, Jean-Baptiste et al

Poster (2012, July 23)

Introduction: Food products represent great biotopes for bacteria. The optimisation of foodstuffs conservation, mattering so economically as from the point of view of the public health, pass by a better ... [more ▼]

Introduction: Food products represent great biotopes for bacteria. The optimisation of foodstuffs conservation, mattering so economically as from the point of view of the public health, pass by a better understanding of those biotopes and their spoilage. Microbiologists had already tried to resolve this problem throughout several approaches. Studies based on classical microbiology cultures were completed by strategies centred on approaches independent from the microbiological culture. Purpose: The current techniques of new generation sequencing give a new dimension to the microbial ecology, through the metagenomic analysis of individuals' large number, within a mixed microbial population. Our aim is to demonstrate that this methodology can be successfully applied to the study of foodstuffs microbial flora, and can be adapted to the specific requirements of food microbiology. Methods: This study was carried out on pork's minced meat and white sausage, with shelf-life tests in various conditions of preservation (temperature and packaging). The rDNA 16S was extracted from the original products and samples in the best-before date and, after standardization, hypervariable regions V5 were sequenced. Results: A total about 130.000 sequences were obtained and a metagenomic analysis succeeded in the taxonomic classification to the genus level for 80 % of this population. The subsequent analysis of microbial populations shows that the majority microbial populations at the expiration date are the same ones which are generally observed during microbiological analysis of these meat products. However, the population subdominants and especially several populations of not cultivable germs were able to be identified. These groups of bacteria, more difficult to obtain by the other methods, must be studied because they participate in the spoilage process of food products. Significance: The sensibility of this technology makes possible the analysis of foodstuffs presenting a very low microbial rate and, thus, allows the identification of the microbial contaminants before they grow the levels detected by cultural methods. [less ▲]

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See detailEtude de la flore bactérienne de steacks tartares prélevés en boucherie, sandwicherie et restaurant par analyse métagénomique ciblée sur l’ADN ribosomial 16S.
Taminiau, Bernard ULg; Delhalle, Laurent ULg; Nezer, Carine et al

Poster (2012, May 24)

Etude de la flore bactérienne de steacks tartares prélevés en boucheries, sandwicheries et restaurants par analyse métagénomique ciblée sur l’ADN ribosomial 16S. Taminiau B.1, Delhalle L 2, Nezer C.2 ... [more ▼]

Etude de la flore bactérienne de steacks tartares prélevés en boucheries, sandwicheries et restaurants par analyse métagénomique ciblée sur l’ADN ribosomial 16S. Taminiau B.1, Delhalle L 2, Nezer C.2, Daube G 1 1 Université de Liège, Faculté de Médecine Vétérinaire, Département des Sciences des Denrées Alimentaires ; Sart Tilman B43 Bis, 4000 Liège, Belgique ; T+32 (0)4 366 40 29 / F+32 (0)4 366 40 44 2 Quality Partner sa, 62 Rue Hayeneux, 4040 Herstal, Belgique ; T+32 (0)4 240 75 00 / F+32 (0)4 240 75 10 Bernard.taminiau@ulg.ac.be Le steak tartare est une préparation à base de viande hachée de boeuf crue assaisonnée et souvent additionnée de condiments. Cette préparation est souvent accompagnée de frites ou étalée dans un sandwich. L’utilisation de viande crue et la composition de cet aliment rend cette préparation très sensible à l’altération d’origine bactérienne. Une meilleure compréhension de la flore bactérienne composant ce produit est indispensable pour contrôler et comprendre les phénomènes d'altération. L'analyse métagénomique ciblée a permis de caractériser les populations bactériennes de plusieurs échantillons de steak tartare achetés dans deux boucheries, deux sandwicheries et deux restaurants et analysés le jour-même. Une analyse classique microbiologique a été réalisée en parallèle. L'analyse métagénomique a été ciblée sur deux régions différentes de l'ADNr 16S bactérien (V&-V3 et V5-V6), afin de comparer l'efficacité d'identification des bactéries présentes. L’analyse métagénomique a permis de collecter un total de 60.500 séquences pour les 6 échantillons analysés par les deux approches et 356 espèces bactériennes différentes ont pu être identifiées via la région V1-V3. Lactobacillus algidus est la principale espèce présente (52% des séquences totales analysées), suivie par Pseudomonas sp. (8,43%) et Photobacterium phosphoreum (7,92%). L'analyse des résultats montre des différences remarquables entre les trois sources commerciales de steak tartare. Les échantillons provenant des deux boucheries et d’un restaurant étaient principalement composés de populations de Lactobacillus et, dans une moindre mesure, de contaminants environnementaux, comme Xanthomonas campestris. Les échantillons provenant de l'un des deux restaurants étaient fortement contaminés par des Leuconostocaceae comme Leuconostoc carnosum ou Weissella sp., ou avec des gamma-protéobactéries comme Pseudomonas sp. ou Psychrobacter sp. Ces derniers échantillons présentaient aussi des signes d’altérations (mauvaise odeur, aspect) qui peuvent ainsi être mis en relation avec la nature et le niveau des populations bactériennes identifiées. Les échantillons provenant des sandwicheries et probablement issues de filières de production industrielles étaient faiblement contaminés mais avec une grande variabilité dans les flores identifiées. Beaucoup d’entre elles ne sont pas classiquement décrites dans les viandes crues et doivent provenir des autres ingrédients. La combinaison du séquençage haut débit couplé à la bioinformatique est désormais un outil puissant pour l’analyse microbiologique des denrées alimentaires. Son application peut être étendue à pratiquement tous les aliments. Enfin, cette technologie ouvre de nouvelles perspectives aux industries agro alimentaires pour améliorer leurs procédés de fabrication, leurs recettes et leurs méthodes de conservation. [less ▲]

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See detailL’analyse métagénomique ciblée au service de la microbiologie des aliments : applications concrètes
Taminiau, Bernard ULg; Nezer, Carine; Delhalle, Laurent ULg et al

Conference (2012, May 23)

La métagénomique est une discipline récente qui s’attache à attribuer une identité, taxonomique, génique ou fonctionnelle, à des fragments d’ADN d’origine inconnue. Son développement et son utilité ... [more ▼]

La métagénomique est une discipline récente qui s’attache à attribuer une identité, taxonomique, génique ou fonctionnelle, à des fragments d’ADN d’origine inconnue. Son développement et son utilité bénéficient grandement de l’essor des techniques de séquençage de seconde génération permettant d’obtenir d’un échantillon de grandes quantités de séquences d’ADN. L’écologie microbienne et la santé intestinale sont les thématiques ayant le plus approfondi la métagénomique et les possibilités qu’elle offre, mais d’autres domaines peuvent grandement bénéficier de cette nouvelle façon d’investiguer les flores microbiennes. Cette étude démontre, à travers plusieurs exemples concrets, que cette méthodologie peut rapidement être adaptée aux exigences spécifiques de la microbiologie des aliments. En effet, les denrées alimentaires périssables, comme la viande et les préparations à base de viande, représentent des biotopes de choix pour les bactéries. L’optimisation de la conservation de ces denrées, importante tant du point de vue économique que de la santé publique, passe par une meilleure connaissance de l’évolution de ces flores et des processus de détérioration des qualités du produit. Les microbiologistes ont depuis longtemps abordé ce problème en utilisant différentes approches dépendantes ou non de la culture microbienne, mais à petite échelle. L’analyse métagénomique est donc une rupture technologique qui allie analyse à grande échelle et large spectre. Un premier exemple a permis de démontrer la capacité de l’analyse métagénomique à identifier les populations bactériennes sous-dominantes dans des produits de viande (viande hachée de porc et boudin blanc). La sensibilité de l’analyse effectuée permet d’identifier les populations microbiennes dès la sortie de production et de comprendre leurs évolutions en fonction de différentes conditions de conservation lors de tests de vieillissement. De plus, la possibilité d’utiliser des échantillons standardisés a servi à comparer plusieurs zones hypervariables de l’ARNr 16S ainsi que d’étudier la reproductibilité de l’analyse. Ensuite, l’impact de la conservation sous-vide de longue durée de la viande bovine a été investigué pour des échantillons d’origines diverses. L’analyse a révélé des différences très marquées malgré un conditionnement apparemment identique. Enfin, nous avons exploré la capacité de cette méthodologie à identifier les bactéries potentiellement responsables d’altérations dans trois matrices alimentaires : le steak tartare, le poisson cru et le fromage. Si l’identification des germes à large spectre est actuellement efficace et reproductible comme nous l’avons démontré ; plusieurs axes de recherche restent à approfondir pour améliorer son utilité dans l’industrie agro-alimentaire. Parmi ceux-ci, il y a la capacité à transposer cette méthodologie pour d’autres micro-organismes comme les moisissures et les champignons, la détermination du degré de profondeur d’analyse utile en fonction du but recherché, et enfin, l’organisation de la manne d’informations qui sont générées par la métagénomique. [less ▲]

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See detailL’extraordinaire potentiel de l’approche métagénomique en microbiologie des aliments: analyse du microbiote de fromages au lait cru et pasteurisé
Delhalle, Laurent ULg; Nezer, Carine; Darcis, Amélie et al

Poster (2012, May 22)

Parmi les techniques culture indépendante, le développement de l’ultra-séquençage a permis de positionner l’analyse métagénomique comme étant la meilleure alternative pour l’étude de microbiotes complexes ... [more ▼]

Parmi les techniques culture indépendante, le développement de l’ultra-séquençage a permis de positionner l’analyse métagénomique comme étant la meilleure alternative pour l’étude de microbiotes complexes. Durant ces trois dernières années, les études métagénomiques ont été consacrées essentiellement à l’analyse d’échantillons environnementaux. Ce travail décrit l’application de cette technique à l’étude des populations bactériennes de quatre types de fromages à pâte molle. Parmi ceux-ci, trois d’entre eux sont des fromages belges typiques à croute lavée (deux sont fabriqués avec du lait cru et le troisième avec du lait pasteurisé). Le quatrième est un fromage français crémeux à base de lait cru. Des analyses microbiologiques classiques et métagénomiques ciblant l’ADNr 16S ont été réalisées dans le cœur et sur la croute des quatre fromages, donnant un total de 8 échantillons. Au total, 48 genres et 163 espèces bactériennes ont été identifiées pour tous les échantillons. Comme attendu, Lactoccocus lactis subsp lactis et/ou cremoris sont les espèces les plus représentées dans le cœur des quatre fromages. Concernant la croute des fromages, les espèces bactériennes les plus abondantes sont Psychrobacter glacinola, Staphylococcus equorum, Corynebacterium casei et Marinilactibacillus psychrotolerans. A noter la présence de Brevibacterium spp et Psychroflexus spp qui permettent d’obtenir la couleur orangée de la croute. Toutes ces espèces sont présentes en différentes proportions suivant l’origine et le processus de fabrication et sont connues pour leurs propriétés technologiques et/ou organoleptiques. Les deux fromages belges au lait cru sont composés de beaucoup d’espèces bactériennes différentes. Tandis que le fromage au lait pasteurisé contient moins d’espèces, principalement Lactococcus lactis (97,6%) dans le cœur. Un résultat inattendu concerne la faible diversité bactérienne du fromage français crémeux à base de lait cru. Seulement deux espèces étaient majoritairement présentes : Lactoccocus lactis subsp cremoris et Leuconostoc citreum avec respectivement 94,9% et 4,9% dans le cœur et 93,8% et 5% dans la croute). Comparé avec les résultats des autres fromages à base de lait cru, ce résultat est particulièrement surprenant. Le microbiote des fromages joue un rôle central sur le processus de fabrication, les qualités organoleptiques et la durée de vie commerciale des produits. L’analyse métagénomique est devenu un outil puissant, rapide et abordable pour identifier, comprendre et maitriser les flores impliquées [less ▲]

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See detailUne nouvelle approche de la microbiologie alimentaire avec l’analyse métagénomique : un exemple avec une analyse de filets de poisson
Delhalle, Laurent ULg; Taminiau, Bernard ULg; Nezer, Carine et al

Poster (2012, May 22)

L’analyse métagénomique est un outil puissant pour étudier la flore bactérienne d’échantillons issus de divers environnements. Cette technologie commence à être appliquée à des aliments mais seulement sur ... [more ▼]

L’analyse métagénomique est un outil puissant pour étudier la flore bactérienne d’échantillons issus de divers environnements. Cette technologie commence à être appliquée à des aliments mais seulement sur l’étude de produits asiatiques fermentés. Ce travail décrit l’application de cette technologie pour étudier la population bactérienne de deux types de filets de poissons : un issu d’un poisson d’eau douce chaude (pangasius) et un de poisson d’eau de mer (églefin). Les échantillons ont été directement analysés le jour de la réception. D’autres échantillons ont été analysés à la fin de leur durée de vie après stockage à 4°C (1/3 de la durée de vie) et 8°C (2/3 de la durée de vie). Ces échantillons ont été conditionnés en barquette de polystyrène et film étirable sous air atmosphérique et en barquette et film PP-EVOH sous atmosphère modifiée (50% N2 / 50% CO2). Des analyses microbiologiques classiques et métagénomiques ciblant l’ADNr 16S ont été réalisés sur tous les échantillons. L’évolution des populations microbiennes des filets de poissons conservés sous différents types de conditionnement et de température a été étudiée. Quarante espèces bactériennes différentes ont été identifiées pour les deux types de poissons. Les bactéries Gram négatives sont majoritaires que ce soit au début ou à la fin de la durée de vie des produits et quel que soit le type de conditionnement. Au début de la conservation, les bactéries Gram négatives présentes sont essentiellement des Moraxellaceae (Acinetobacter spp, Psychrobacter sp.), Pseudomonadaceae (Pseudomonas spp), et Shewanella spp et les bactéries Gram positives sont des Lactobacillaceae (Carnobacterium spp), Brochothrix thermosphacta et Planococcus donghaensis (seulement pour le pangasius). Suivant le type de conditionnement et le type de poisson, des variations importantes de la flore initiale ont été observées. La croissance de certaines espèces Gram négatives pourrait être un indicateur d’altération. Par conséquent, l’analyse métagénomique pourrait être un outil supplémentaire pour fixer adéquatement la date limite de consommation. Pour le pangasius, Planococcus donghaensis est présent uniquement avant le conditionnement et sa présence pourrait être un indicateur de la fraîcheur du poisson. Cette étude a permis d’appliquer l’analyse métagénomique pour identifier et mesurer les proportions relatives des espèces bactériennes dans les filets de poissons au cours du temps. [less ▲]

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See detailStudy of the microbial diversity in vacuum-packed chilled beef from different origins through a metagenomics approach
Didimo Imazaki, Pedro Henrique ULg; Taminiau, Bernard ULg; Nezer, Carine et al

Poster (2012, April)

Despite a diverse initial microbial population, bacterial spoilage of vacuum-packed chilled beef is mainly due to the growth of psychrotrophic bacteria. The study of the microflora of vacuum-packed ... [more ▼]

Despite a diverse initial microbial population, bacterial spoilage of vacuum-packed chilled beef is mainly due to the growth of psychrotrophic bacteria. The study of the microflora of vacuum-packed chilled beef remains a challenge since some members of the microflora may be missed or not identified by cultivation-based methods. The aim of this study was to evaluate the microbial diversity in eight batches of vacuum-packed chilled beef from different origins (Australia, Belgium, Brazil, Ireland and United Kingdom) by metagenomics. Longissimus dorsi muscle samples were homogenized and analysed in early and late stages of their shelf life by metagenomics. The metagenomic assays consisted in DNA extraction, 16S ribosomal RNA gene amplification, pyrosequencing and data analysis. All samples, except for two batches from Australia, presented a high microbial diversity in the beginning of their shelf life. Enterobacteriaceae, Pseudomonas, Burkholderia, Lactobacillus and Sterotrophomonas were some of the major bacteria identified at this stage of storage. The dominant flora (> 80 % of relative abundance) in two Australian batches was composed by Carnobacterium. At the end of the shelf life of the samples, a decrease in microbial diversity was observed in almost all batches. At this stage of storage, Carnobacterium, Lactobacillus, Lactococcus and Enterococcus were some of the major genera identified. Carnobacterium remained the dominant flora in the two Australian batches cited above, which could explain the long shelf life applicable to this meat (140 days) as some Carnobacterium strains induce a biopreservative effect especially by producing bacteriocins with a wide inhibition spectrum. Metagenomics showed to be a very useful tool to study the microbial population of a complex matrix such as meat since some of the identified genera such as Lactobacillus and Carnobacterium are known not to grow or to grow slowly in media commonly used for the isolation and cultivation of total viable counts. [less ▲]

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See detailNeuroimmune connections in ovine pharyngeal tonsil: potential site for prion neuroinvasion
Toppets, Vinciane ULg; Piret, Joëlle ULg; Kirschvink, Nathalie et al

in Cell & Tissue Research (2012)

Recent studies have proved the possible implication of nasal associated lymphoid tissues, mainly the pharyngeal tonsil, in prion pathogenesis. However, the mechanisms of this neuroinvasion are still being ... [more ▼]

Recent studies have proved the possible implication of nasal associated lymphoid tissues, mainly the pharyngeal tonsil, in prion pathogenesis. However, the mechanisms of this neuroinvasion are still being debated. To determine the potential sites for prion neuroinvasion inside the ovine pharyngeal tonsil, the topography of neurofilaments heavy (200 kDa) (NFH), neurofilaments light (70 kDa) (NFL) and glial fibrillar acidic protein (GFAP) was semi-quantitatively analysed inside the different compartments of the tonsil. The results showed that the most innervated areas were the interfollicular area and the connective tissue located beneath the respiratory epithelium. Even if the germinal centre of the lymphoid follicles was poorly innervated, the existence of rare follicular dendritic cell-nerve synapses inside the germinal centre indicates that this mechanism of neuroinvasion is possible but unlikely to be unique. The host PRNP genotype did not influence the pattern of innervation in these different tonsil compartments, unlike age: an increase of nerve endings in a zone of high trafficking cells beneath the respiratory epithelium occurred with ageing. A minimal age-related increase of innervation inside the lymphoid follicles was also observed. An increase in nerve fibre density around the lymphoid follicles, in an area rich in mobile cells able to transport PrPd, could ensure a more efficient infectivity, not in the early phase but in the advanced phase of lymphoinvasion after amplification of PrPd, or could act as direct site of entry during neuroinvasion. [less ▲]

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See detailA Review of Known and Hypothetical Transmission Routes for Noroviruses
Mathijs, E.; Stals, A.; Baert, L. et al

in Food and Environmental Virology (2012), 4(4), 131-152

Human noroviruses (NoVs) are considered a worldwide leading cause of acute non-bacterial gastroenteritis. Due to a combination of prolonged shedding of high virus levels in feces, virus particle shedding ... [more ▼]

Human noroviruses (NoVs) are considered a worldwide leading cause of acute non-bacterial gastroenteritis. Due to a combination of prolonged shedding of high virus levels in feces, virus particle shedding during asymptomatic infections, and a high environmental persistence, NoVs are easily transmitted pathogens. Norovirus (NoV) outbreaks have often been reported and tend to affect a lot of people. NoV is spread via feces and vomit, but this NoV spread can occur through several transmission routes. While person-to-person transmission is without a doubt the dominant transmission route, human infective NoV outbreaks are often initiated by contaminated food or water. Zoonotic transmission of NoV has been investigated, but has thus far not been demonstrated. The presented review aims to give an overview of these NoV transmission routes. Regarding NoV person-to-person transmission, the NoV GII. 4 genotype is discussed in the current review as it has been very successful for several decades but reasons for its success have only recently been suggested. Both pre-harvest and post-harvest contamination of food products can lead to NoV food borne illness. Pre-harvest contamination of food products mainly occurs via contact with polluted irrigation water in case of fresh produce or with contaminated harvesting water in case of bivalve molluscan shellfish. On the other hand, an infected food handler is considered as a major cause of post-harvest contamination of food products. Both transmission routes are reviewed by a summary of described NoV food borne outbreaks between 2000 and 2010. A third NoV transmission route occurs via water and the spread of NoV via river water, ground water, and surface water is reviewed. Finally, although zoonotic transmission remains hypothetical, a summary on the bovine and porcine NoV presence observed in animals is given and the presence of human infective NoV in animals is discussed. © 2012 Springer Science+Business Media New York. [less ▲]

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See detailRetrospective analysis of a listeria monocytogenes contamination episode in raw milk goat cheese using quantitative microbial risk assessment tools
Delhalle, Laurent ULg; Ellouze, M.; Yde, M. et al

in Journal of Food Protection (2012), 75(12), 2122-2135

In 2005, the Belgian authorities reported a Listeria monocytogenes contamination episode in cheese made from raw goat's milk. The presence of an asymptomatic shedder goat in the herd caused this ... [more ▼]

In 2005, the Belgian authorities reported a Listeria monocytogenes contamination episode in cheese made from raw goat's milk. The presence of an asymptomatic shedder goat in the herd caused this contamination. On the basis of data collected at the time of the episode, a retrospective study was performed using an exposure assessment model covering the production chain from the milking of goats up to delivery of cheese to the market. Predictive microbiology models were used to simulate the growth of L. monocytogenes during the cheese process in relation with temperature, pH, and water activity. The model showed significant growth of L. monocytogenes during chilling and storage of the milk collected the day before the cheese production (median increase of 2.2 log CFU/ml) and during the addition of starter and rennet to milk (median increase of 1.2 log CFU/ml). The L. monocytogenes concentration in the fresh unripened cheese was estimated to be 3.8 log CFU/g (median). This result is consistent with the number of L. monocytogenes in the fresh cheese (3.6 log CFU/g) reported during the cheese contamination episode. A variance-based method sensitivity analysis identified the most important factors impacting the cheese contamination, and a scenario analysis then evaluated several options for risk mitigation. Thus, by using quantitative microbial risk assessment tools, this study provides reliable information to identify and control critical steps in a local production chain of cheese made from raw goat's milk. © International Association for Food Protection. [less ▲]

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See detailMolecular Detection and Genotyping of Noroviruses
Stals, A.; Mathijs, E.; Baert, L. et al

in Food and Environmental Virology (2012), 4(4), 153-167

Noroviruses (NoVs) are a major cause of gastroenteritis worldwide in humans and animals and are known as very infectious viral agents. They are spread through feces and vomit via several transmission ... [more ▼]

Noroviruses (NoVs) are a major cause of gastroenteritis worldwide in humans and animals and are known as very infectious viral agents. They are spread through feces and vomit via several transmission routes involving person-to-person contact, food, and water. Investigation of these transmission routes requires sensitive methods for detection of NoVs. As NoVs cannot be cultivated to date, detection of these viruses relies on the use of molecular methods such as (real-time) reverse transcriptase polymerase chain reaction (RT-PCR). Regardless of the matrix, detection of NoVs generally requires three subsequent steps: a virus extraction step, RNA purification, and molecular detection of the purified RNA, occasionally followed by molecular genotyping. The current review mainly focused on the molecular detection and genotyping of NoVs. The most conserved region in the genome of human infective NoVs is the ORF1/ORF2 junction and has been used as a preferred target region for molecular detection of NoVs by methods such as (real-time) RT-PCR, NASBA, and LAMP. In case of animal NoVs, broad range molecular assays have most frequently been applied for molecular detection. Regarding genotyping of NoVs, five regions situated in the polymerase and capsid genes have been used for conventional RT-PCR amplification and sequencing. As the expected levels of NoVs on food and in water are very low and inhibition of molecular methods can occur in these matrices, quality control including adequate positive and negative controls is an essential part of NoV detection. Although the development of molecular methods for NoV detection has certainly aided in the understanding of NoV transmission, it has also led to new problems such as the question whether low levels of human NoV detected on fresh produce and shellfish could pose a threat to public health. © 2012 Springer Science+Business Media New York. [less ▲]

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See detailValidation of a method for simultaneous isolation of shiga toxin-producing Escherichia coli O26, O103, O111, and O145 from minced beef by an international ring-trial
Verstraete, K.; De Zutter, L.; Robyn, J. et al

in Foodborne Pathogens and Disease (2012), 9(5), 412-417

An isolation method described by Possé et al. (FEMS Microbiol Lett 2008;282:124-131) was satisfactorily validated in an international ring-trial using artificially contaminated minced beef samples. Until ... [more ▼]

An isolation method described by Possé et al. (FEMS Microbiol Lett 2008;282:124-131) was satisfactorily validated in an international ring-trial using artificially contaminated minced beef samples. Until now, no validated method existed for the simultaneous isolation of Shiga toxin-producing Escherichia coli serogroups O26, O103, O111, and O145 in food. Twelve laboratories from five European countries participated and received 16 inoculated beef samples contaminated with cold-stressed cells of the four serogroups O26, O103, O111, and O145 in two levels (approximately 30 and 300 CFU 25g-1) in duplicate. In addition, they received four non-inoculated samples. The isolation protocol comprised a selective enrichment step, a selective isolation step on a non-O157 agar plate differentiating the serogroups by color, followed by confirmation by plating on confirmation agar media and agglutination. All laboratories were able to isolate the inoculated serogroups from the samples, both for the high and the low inoculation level. Results did not differ whether in-house-prepared or ready-to-use non-O157 agar plates were used, demonstrating that by following the instructions laboratories managed to perform the complete protocol with success. © Copyright 2012, Mary Ann Liebert, Inc. [less ▲]

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See detailComplete genome sequence of a novel bovine norovirus: Evidence for slow genetic evolution in genogroup III genotype 2 noroviruses
Mauroy, Axel ULg; Scipioni, A.; Mathijs, E. et al

in Journal of Virology (2012), 86(22), 12449-12450

A new genogroup III genotype 2 bovine norovirus, B309/2003/BE, was entirely sequenced and genetically compared to the original Newbury2/1976/UK strain and to Dumfries/1994/UK, detected in 1976 and 1994 ... [more ▼]

A new genogroup III genotype 2 bovine norovirus, B309/2003/BE, was entirely sequenced and genetically compared to the original Newbury2/1976/UK strain and to Dumfries/1994/UK, detected in 1976 and 1994, respectively. Interestingly, except in welldefined coding regions (N-terminal protein, 3A-like protease, hypervariable region of the capsid protein, and C-terminal part of the minor structural protein), very low genetic differences were noted between the entire genomes of these three strains along a 30-year-long period. It allowed some hypotheses of hotspots of genetic evolution through a low genetic evolution background in genotype 2 genogroup III bovine noroviruses. © 2012, American Society for Microbiology. [less ▲]

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