References of "Nezer, Carine"
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See detailHigh throughput sequencing analysis reveals genetic variability and selection pressure in different murine norovirus genomic regions during in vitro replication
Mauroy, Axel ULg; Taminiau, Bernard ULg; Nezer, Carine et al

Poster (2014, July)

Murine norovirus (MuNoV), a single stranded positive sense RNA virus belonging to the Caliciviridae family, is considered as a representative model for human norovirus infections, one of the most ... [more ▼]

Murine norovirus (MuNoV), a single stranded positive sense RNA virus belonging to the Caliciviridae family, is considered as a representative model for human norovirus infections, one of the most important etiological cause of both epidemic and sporadic gastroenteritis cases worldwide. Four open reading frames are described into its genome: ORF1 codes the non-structural (NS) proteins, including the viral RNA dependent RNA polymerase (RdRp); ORF2 codes the single capsid protein (VP1), wherein two domains are present: a relatively conserved domain (“shell”) and a more variable domain (“protruding”); ORF3 codes a minor structural protein; and ORF4, currently only found in viruses genetically related to MuNoV codes a virulence factor. In this study, we demonstrated by high throughput sequencing that, during serial passages of MuNoV in cell culture, the substitution rates, estimated by Bayesian inferences, did not significantly differ across the five targeted genomic regions except one. These rates were similar in four genomic regions encompassing partial non-structural 1-2 protein (NS1-2)-, NS5-, NS6-, NS7 (RdRp)- and VP1-coding sequences (coding the conserved part of the protein also including the ORF4 region). In the partial minor structural protein-coding region, this substitution rate was however estimated to be at least one log higher when expressed as substitution/site/day. The precise localisation of the detected nucleotide point mutations (substitution, deletion and insertion) were reported as well as the quantitative increase or decrease of the sequences harbouring them along ten cell culture passages. The non-silent amino acid mutations were also depicted in 3D models for four out of the five studied regions. These results have important implications for different norovirus research fields, especially in terms of diagnosis, classification methodology and genetic evolution. [less ▲]

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See detailStudy of the microbial flora of steak tartare by metagenomic approach
Korsak Koulagenko, Nicolas ULg; Delhalle, Laurent ULg; Nezer, Carine et al

Poster (2014, May 06)

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See detailL'analyse par séquençage à haut débit révèle la variabilité génétique et la pression de sélection dans différentes régions génomiques du norovirus murin durant sa réplication in vitro
Mauroy, Axel ULg; Taminiau, Bernard ULg; Nezer, Carine et al

Conference (2014, March)

Le norovirus murin (MuNoV), un virus à ARN de polarité positive appartenant à la famille des Caliciviridae, est considéré comme un modèle adéquat pour les infections humaines à norovirus, une des causes ... [more ▼]

Le norovirus murin (MuNoV), un virus à ARN de polarité positive appartenant à la famille des Caliciviridae, est considéré comme un modèle adéquat pour les infections humaines à norovirus, une des causes étiologiques les plus importantes dans les cas de gastroentérite épidémique ou sporadique dans le monde entier. Quatre cadres de lecture ouverts (ORF) sont décrits au sein de son génome : l’ORF1 code les protéine non structurales (NS), dont l’ARN polymérase ARN dépendante virale (RdRp) ; l’ORF2 code l’unique protéine de capside (VP1), dans laquelle sont décrites deux régions : une relativement conservée (domaine « shell ») et une autre beaucoup plus variable (domaine « protruding ») ; l’ORF3 code une protéine structurale mineure ; et l’ORF4, actuellement uniquement décrit chez les virus génétiquement apparentés au MuNoV, code un facteur de virulence. Dans cette étude, nous démontrons par séquençage à haut débit que, durant des passages successifs du MuNoV en culture cellulaire, les taux de substitution, estimés par inférences Bayésiennes, n’ont pas significativement différé au travers des cinq régions génomiques ciblées à l’exception d’une région bien précise. Ces taux étaient similaires pour quatre régions englobant des séquences partielles codant les protéines non structurales NS1-2, NS5, NS6 et NS7 (RdRp) et VP1 dans sa région conservée (incluant également l’ORF4). Dans la région codant partiellement la protéine structurale mineure, ce taux de substitution, exprimé en substitution/site/jour, a été cependant estimé être plus élevée d’au moins une unité logarithmique. La localisation précise des mutations ponctuelles détectées (substitution, délétion et insertion) est rapportée ainsi que l’augmentation ou la diminution quantitative du nombre des séquences qui les présentaient au cours de dix passages successifs en culture cellulaire. Les localisations des mutations non silencieuses ont aussi été représentées dans une modélisation tridimensionnelle de quatre des cinq régions étudiées. Ces résultats ont d’importantes implications pour différents champs de recherche sur les norovirus, spécialement en termes de diagnostic, de méthodologie de classification et d’évolution génétique. [less ▲]

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See detailMicrobiota characterization of a protected designation of origin Belgian cheese: Herve cheese, using metagenomic analysis.
Delcenserie, Véronique ULg; Taminiau, Bernard ULg; Delhalle, Laurent ULg et al

in Journal of Dairy Science (2014), 97

Herve cheese is a Belgian soft cheese with a washed rind, and is made from raw or pasteurized milk. The specific microbiota of this cheese has never previously been fully explored and the use of raw or ... [more ▼]

Herve cheese is a Belgian soft cheese with a washed rind, and is made from raw or pasteurized milk. The specific microbiota of this cheese has never previously been fully explored and the use of raw or pasteurized milk in addition to starters is assumed to affect the microbiota of the rind and the heart. The aim of the study was to analyze the bacterial microbiota of Herve cheese using classical microbiology and a metagenomic approach based on 16S ribosomal DNA pyrosequencing. Using classical microbiology, the total counts of bacteria were comparable for the 11 samples of tested raw and pasteurized milk cheeses, reaching almost 8 log cfu/g. Using the metagenomic approach, 207 different phylotypes were identified. The rind of both the raw and pasteurized milk cheeses was found to be highly diversified. However, 96.3 and 97.9% of the total microbiota of the raw milk and pasteurized cheese rind, respectively, were composed of species present in both types of cheese, such as Corynebacterium casei, Psychrobacter spp., Lactococcus lactis ssp. cremoris, Staphylococcus equorum, Vagococcus salmoninarum, and other species present at levels below 5%. Brevibacterium linens were present at low levels (0.5 and 1.6%, respectively) on the rind of both the raw and the pasteurized milk cheeses, even though this bacterium had been inoculated during the manufacturing process. Interestingly, Psychroflexus casei, also described as giving a red smear to Raclettetype cheese, was identified in small proportions in the composition of the rind of both the raw and pasteurized milk cheeses (0.17 and 0.5%, respectively). In the heart of the cheeses, the common species of bacteria reached more than 99%. The main species identified were Lactococcus lactis ssp. cremoris, Psychrobacter spp., and Staphylococcus equorum ssp. equorum. Interestingly, 93 phylotypes were present only in the raw milk cheeses and 29 only in the pasteurized milk cheeses, showing the high diversity of the microbiota. Corynebacterium casei and Enterococcus faecalis were more prevalent in the raw milk cheeses, whereas Psychrobacter celer was present in the pasteurized milk cheeses. However, this specific microbiota represented a low proportion of the cheese microbiota. This study demonstrated that Herve cheese microbiota is rich and that pasteurized milk cheeses are microbiologically very close to raw milk cheeses, probably due to the similar manufacturing process. The characterization of the microbiota of this particular protected designation of origin cheese was useful in enabling us to gain a better knowledge of the bacteria responsible for the character of this cheese. [less ▲]

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See detailImpact of agricultural practices on soil microbial communities in Belgium
Degrune, Florine ULg; Taminiau, Bernard ULg; Dufrêne, Marc ULg et al

Poster (2013, December 11)

The use of fertilizers in agricultural soils is becoming a real environmental issue (an obvious example is eutrophication caused by leaching of phosphorus and nitrates). Much research has focused on ... [more ▼]

The use of fertilizers in agricultural soils is becoming a real environmental issue (an obvious example is eutrophication caused by leaching of phosphorus and nitrates). Much research has focused on finding ways to reduce the use of chemicals, and investigating microbial life may lead to solutions. We know that bacteria and fungi are deeply involved in nutrient cycles. Recently the emergence of massive parallel sequencing has enabled us to realize that microbial diversity is huger than we expected. With such a tool it should be possible to study how soil management practices affect the microbial diversity of agricultural soils. A few such studies have been conducted, most of them focusing on bacteria. For Belgium in particular, there is a lack of data on this topic. Here the aim was to see how residue management and tillage practices affect communities of both bacteria and fungi in Belgian agricultural soils. For this we used 454 pyrosequencing of 16S bacterial and 28S fungal rRNA genes. Soil samples came from an experiment in which faba beans were grown with four soil management practices (tillage and no tillage, with and without crop residues), each repeated four times in a Latin square. Several chemical and physical characteristics were measured on each sample. The results show that fungi and bacteria are both impacted by Tillage practices. The main soil drivers are Magnesium and Phosphorus for Fungi communities, and Phosphorus and Potassium for bacteria communities. Finally, the fungi variance observed between plots is explained at 38% by Tillage, Magnesium and phosphorus. And the bacteria variance is explained at 28% by Tillage, Phosphorus and Potassium. [less ▲]

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See detailEvaluation of the bacterial diversity and its evolution during storage of fresh beef from British and Belgian origins under different atmosphere and temperature conditions
Didimo Imazaki, Pedro Henrique ULg; Tahiri, Assia ULg; Taminiau, Bernard ULg et al

Conference (2013, December 10)

The purpose of this study was to evaluate the bacterial diversity and its evolution during storage of fresh beef, depending on its origin, packaging and storage temperature, by using a metagenomic ... [more ▼]

The purpose of this study was to evaluate the bacterial diversity and its evolution during storage of fresh beef, depending on its origin, packaging and storage temperature, by using a metagenomic approach. Metagenomic analyzes revealed that the origin, atmosphere and temperature conditions influenced the selection of the predominant flora. Vacuum-packaged British samples presented higher concentrations of Lactobacillus algidus at the begging of the experiment than Belgian samples. Furthermore, the development of Lactobacillus algidus was favored in British and Belgian samples preserved under vacuum at −1 °C, while a predominance of Lactococcus piscium was observed for samples stored at +4 °C. These microorganisms have already been isolated from beef, but taking into account that the knowledge about these two species is currently limited, it is still not possible to state if the conservability of the tested samples was influenced by the presence of these bacteria. Moreover, storage under modified atmosphere favored the development of Leuconostoc gasicomitatum in both British and Belgian samples. This specie is often associated with spoilage of cold-stored modified-atmosphere-packaged (MAP) nutrient-rich foods. This result can partially explain the short shelf-life of the samples once they are stored under this condition. Metagenomics showed to be a useful tool to study the microbial population of a complex matrix since some of the identified genera could not have grown or have grown slowly in culture media commonly used. In addition, it helped to clarify the evolution of the bacterial ecosystem associated to meat during its storage. [less ▲]

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See detailBacterial diversity and its evolution during storage of fresh beef from different origins under different atmosphere and temperature conditions
Didimo Imazaki, Pedro Henrique ULg; Tahiri, Assia ULg; Taminiau, Bernard ULg et al

Poster (2013, October 11)

The purpose of this study was to evaluate the bacterial diversity and its evolution during storage of fresh beef, depending on its origin, packaging and storage temperature. Two batches of three vacuum ... [more ▼]

The purpose of this study was to evaluate the bacterial diversity and its evolution during storage of fresh beef, depending on its origin, packaging and storage temperature. Two batches of three vacuum packed striploins from United Kingdom and Belgium were obtained from a food wholesaler located in the Walloon Region. Fifteen days after slaughter, the striploins were sliced and individually kept under vacuum for 30 days: i) at −1 °C; ii) at +4 °C and iii) at −1 °C for 15 days and then at +4 °C for 15 days. The bacterial diversity was evaluated by metagenomic approach 15, 30 and 45 days after slaughter. Furthermore, each 15 days part of the vacuum packed striploin slices were repacked under modified atmosphere (70 % O2/30 % CO2), stored at +4 °C for 2 days and at +8 °C for 5 days, and then analyzed. Metagenomic analysis revealed a selection of the initial flora depending on atmosphere and temperature conditions. The development of Lactobacillus algidus was favored in samples preserved under vacuum at −1 °C, while a predominance of Lactococcus piscium was observed for samples stored at +4 °C. Moreover, storage under modified atmosphere favored the development of Leuconostoc gasicomitatum. These microorganisms have already been isolated from beef, but no study has evaluated their role in food conservation. The next step of this study will be to isolate and characterize strains of Lactobacillus algidus from meat and to assess their bioprotective potential. [less ▲]

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See detailLa diversité bactérienne et son évolution pendant la conservation de viandes bovines fraîches de différentes origines conditionnées sous vide
Didimo Imazaki, Pedro Henrique ULg; Tahiri, Assia ULg; Taminiau, Bernard ULg et al

Poster (2012, December)

Le but de cette étude à été d'évaluer la diversité bactérienne et son évolution pendant la conservation de viandes bovine fraîches sous vide, en fonction de leur origine et du respect ou non d’une ... [more ▼]

Le but de cette étude à été d'évaluer la diversité bactérienne et son évolution pendant la conservation de viandes bovine fraîches sous vide, en fonction de leur origine et du respect ou non d’une température proche du point de congélation. Les dénombrements réalisés ont mis en évidence que les viandes d’origines britannique et belge testées présentent un écosystème microbien différent. Les analyses par approche métagénomique permettront d’éclaircir ces différences, surtout en ce qui concerne la présence de bactéries pouvant jouer un rôle "bioprotecteur" permettant d’améliorer la conservabilité des viandes. [less ▲]

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See detailValidation de la qualité de la viande hachée de porc par une approche métagénomique ciblée
Taminiau, Bernard ULg; Nezer, Carine; Adolphe, Ysabelle ULg et al

Conference (2012, November 13)

Food products represent great biotopes for bacteria. The optimization of foodstuffs conservation pass by a better understanding of those biotopes and their spoilage. The current techniques of new ... [more ▼]

Food products represent great biotopes for bacteria. The optimization of foodstuffs conservation pass by a better understanding of those biotopes and their spoilage. 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 validation of the quality of foodstuffs during storage. This study was carried out on pork minced meat with shelf-life tests in various conditions of preservation (temperature and packaging). The analysis was performed in parallel with standardized microbiological methods and with massive sequencing of two hypervariables regions of the rDNA 16S. The results show an excellent correlation between the two approaches and underline the tremendous utility of metagenomic analysis for in-depth characterization of the potential altering bacteria in fresh meat. [less ▲]

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See detailViandes bovines à longue durée de conservation conditionnées sous vide : isolement et caractérisation de souches de Carnobacterium
Didimo Imazaki, Pedro Henrique ULg; Tahiri, Assia ULg; Baptista Rodrigues, Ana Lucia ULg et al

Poster (2012, November 13)

The lactic acid bacteria Carnobacterium divergens and Carnobacterium maltaromaticum are often associated to meat and meat products and may be used as a protective culture, improving the microbial ... [more ▼]

The lactic acid bacteria Carnobacterium divergens and Carnobacterium maltaromaticum are often associated to meat and meat products and may be used as a protective culture, improving the microbial stability and the safety of these products. In this context, the aim of this study was to isolate and characterize Carnobacterium from long shelf-life vacuum-packed beef. LAB counts after culture at +22°C remained below 2.0 log UFC/cm², even at the end of shelf life. On the other hand, the ecosystem evaluation performed by metagenomics revealed the predominance of Carnobacterium and Lactobacillus on the samples. After spreading of a peptone water suspension obtained from the samples on PCA, pure isolates were collected and identified by API 50 CHL galleries. Seventy-eight % of isolates were C. maltaromaticum, 3 % C. divergens and 19 % could not be identified. The next step of this work will consist in performing a genotypic and functional characterization of these Carnobacterium isolates. [less ▲]

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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 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, September 21)

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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 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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