Cartography of the microbial diversity from French PDO milks and cheeses and investigation of the technological drivers

International audiencePDO cheeses are generally considered as high quality, non-standardised products whose sensory richness arises from a variety of milk production and processing conditions. These practices would contribute to shaping microbial biodiversity and to the selection of ecotypes of environmental species that have adapted to the dairy environment. We seek to demonstrate this hypothesis by characterising the biotic and technological drivers of the cheese microbiota across the French PDO production areas. As a beneficiary of France Genomique major sequencing projects in 2017, MetaPDOcheese is the first large-scale study of the microbiota in all French PDO cheeses.Massive sequencing of "taxonomic marker" gene amplicons was used to describe the bacterial and fungal diversity of 1,145 cheeses and 390 milks originating from 44 PDO areas. A database consolidating all the technical information associated with the samples was also created. Up to 289 bacterial genera and 820 species, as well as 175 fungal genera and 333 species were identified from > 1 billion sequences generated from the 2,306 cheese samples (cores and rinds). Up to 549 genera and 1,230 bacterial species, as well as 671 genera and 1,367 fungal species were identified from > 180 million sequences generated from the milk samples. A core microbiome comprising several tens of bacterial and fungal taxa was identified in milk. The milk’s microbiota differed according to the dairy species. The core microbiome in cheese was limited to one fungal species (Geotrichum candidum) at 100% prevalence. A core microbiome was identified across each cheese family, mainly composed of species potentially added as starters for cheese making and ripening. The cheese microbiota differed in terms of community richness and composition between the 7 cheese families and within families, according to PDO labels. Secondary structuring factors, such as the ripening duration, were also highlighted across each cheese family

MetaPDOcheese : Cartographie de la diversité microbienne des laits et des fromages AOP français et exploration des déterminants technologiques

National audienceLes fromages d'appellation d'origine protégée (AOP) sont généralement considérés comme des produits fermentés non standardisés de haute qualité dont la richesse sensorielle résulte de la diversité des conditions de production et de transformation du lait. Dans cette étude, l’objectif est de caractériser la diversité des communautés microbiennes des fromages et des laits associés, et d’identifier les facteurs de leur structuration en lien avec la biogéographie, la gestion des troupeaux et les pratiques de transformation fromagère. Grâce à la contribution des filières AOP, 1 145 fromages AOP (croûte et cœur) et 370 laits associés ont été échantillonnés couvrant la diversité des 44 fromages AOP affinés français. L’ampleur de l’échantillonnage des laits et des fromages et la caractérisation de leurs communautés bactériennes et fongiques par métagénétique, a permis de générer un grand jeu de données précieux car il est associé à des métadonnées sensibles fournies par les filières portant sur les pratiques de production utilisées pour chaque fromage testé. Au total, 1 230 espèces bactériennes et 1 367 espèces fongiques ont été identifiées dans les échantillons de lait, avec des variations en fonction de l'espèce laitière. Dans les fromages, 820 espèces bactériennes et 333 espèces fongiques ont été identifiées. Le core microbiome du fromage est limité à une espèce fongique (Geotrichum candidum) avec une prévalence de 100 %. Le microbiote du fromage diffère en termes de richesse et de composition entre les sept familles de fromage et au sein des familles, en fonction des labels AOP. Des facteurs structurants secondaires, tels que l'espèce laitière, la zone géographique et les pratiques d'affinage du fromage, ont également été mis en évidence entre les familles de fromages, démontrant une contribution de la biogéographie et du savoir-faire spécifique à l'AOP dans la formation du microbiote du fromage

Recovery of partial 16S rDNA sequences suggests the presence of Crenarchaeota in the human digestive ecosystem

Human feces collected from 10 healthy teenagers was analyzed for the presence of Crenarchaeota. After a first polymerase chain reaction (PCR) with Archaea-specific primers, a nested real-time PCR was performed using Crenarchaeota-specific primers. Real-time Crenarchaeotal PCR products detected from four subjects were cloned and the sequencing revealed that most of the partial 16S rRNA gene sequences were highly similar (≥97% homology) to sequences affiliated to the Sulfolobus group of the Crenarchaeota phylum. Our findings suggest for the first time that Crenarchaeota might be present in the microbiota of the human digestive ecosystem in which this phylum has never been found yet

Monitoring Bacterial Communities in Raw Milk and Cheese by Culture-Dependent and -Independent 16S rRNA Gene-Based Analyses

The diversity and dynamics of bacterial populations in Saint-Nectaire, a raw-milk, semihard cheese, were investigated using a dual culture-dependent and direct molecular approach combining single-strand conformation polymorphism (SSCP) fingerprinting and sequencing of 16S rRNA genes. The dominant clones, among 125 16S rRNA genes isolated from milk, belonged to members of the Firmicutes (58% of the total clones) affiliated mainly with the orders Clostridiales and the Lactobacillales, followed by the phyla Proteobacteria (21.6%), Actinobacteria (16.8%), and Bacteroidetes (4%). Sequencing the 16S rRNA genes of 126 milk isolates collected from four culture media revealed the presence of 36 different species showing a wider diversity in the Gammaproteobacteria phylum and Staphylococcus genus than that found among clones. In cheese, a total of 21 species were obtained from 170 isolates, with dominant species belonging to the Lactobacillales and subdominant species affiliated with the Actinobacteria, Bacteroidetes (Chryseobacterium sp.), or Gammaproteobacteria (Stenotrophomonas sp.). Fingerprinting DNA isolated from milk by SSCP analysis yielded complex patterns, whereas analyzing DNA isolated from cheese resulted in patterns composed of a single peak which corresponded to that of lactic acid bacteria. SSCP fingerprinting of mixtures of all colonies harvested from plate count agar supplemented with crystal violet and vancomycin showed good potential for monitoring the subdominant Proteobacteria and Bacteroidetes (Flavobacteria) organisms in milk and cheese. Likewise, analyzing culturable subcommunities from cheese-ripening bacterial medium permitted assessment of the diversity of halotolerant Actinobacteria and Staphylococcus organisms. Direct and culture-dependent approaches produced complementary information, thus generating a more accurate view of milk and cheese microbial ecology

Caenorhabditis elegans, un modèle utilisé pour le criblage de microbiotes fromagers à effet santé

International audienceCaenorhabditis elegans, un modèle utilisé pour le criblage de microbiotes fromagers à effet sant

Lactic Starter Dose Shapes S. aureus and STEC O26:H11 Growth, and Bacterial Community Patterns in Raw Milk Uncooked Pressed Cheeses

International audienceAdding massive amounts of lactic starters to raw milk to manage the sanitary risk in the cheese-making process could be detrimental to microbial diversity. Adjusting the amount of the lactic starter used could be a key to manage these adverse impacts. In uncooked pressed cheeses, we investigated the impacts of varying the doses of a lactic starter (the recommended one, 1×, a 0.1× lower and a 2× higher) on acidification, growth of Staphylococcus aureus SA15 and Shiga-toxin-producing Escherichia coli (STEC) O26:H11 F43368, as well as on the bacterial community patterns. We observed a delayed acidification and an increase in the levels of pathogens with the 0.1× dose. This dose was associated with increased richness and evenness of cheese bacterial community and higher relative abundance of potential opportunistic bacteria or desirable species involved in cheese production. No effect of the increased lactic starter dose was observed. Given that sanitary criteria were paramount to our study, the increase in the pathogen levels observed at the 0.1× dose justified proscribing such a reduction in the tested cheese-making process. Despite this, the effects of adjusting the lactic starter dose on the balance of microbial populations of potential interest for cheese production deserve an in-depth evaluation

Cadaverine, putrescine, and histamine formation of Morganella morganii in raclette-type cheese

International audienceThe influence of Morganella morganii isolated from cheese on the formation of biogenic amines was studied in raclette-type cheeses. Three variants were produced. One variant containing a cadaverine-and histamine-forming strain, one variant with a putrescine-and histamine-forming strain, and a variant without M. morganii. After 130 d of ripening, live M. morganii was found in the outer layers but no longer inside the cheese. The cheeses with the cadaverine-forming strain exhibited a decreasing cadaverine gradient from the outside (on average 310 mg kg(-1)) to the inside (160 mg kg(-1)). Putrescine was present in the cheeses with the putrescine-forming strain. Its concentration averaged 59 mg kg(-1) in all layers. All cheeses with M. morganii contained also histamine with concentration less than 50 mg kg(-1). The results reveal new information on the survival of M. morganii as well as its ability to form biogenic amines in cheese

Biodiversity and factors of variation of microbial flora on the teats of dairy cows

International audienceTo determine how farming practices and the intrinsic characteristics of individual dairy cows can influence the composition of the microbial community on teat skin, sixteen dairy farms in the Cantal region of France were studied. Microbial groups of the teat skin of 6 dairy cows per farm were counted using 10 dairy-specific media. Farms were classed into three groups according to the milking hygiene practices, the litter and the forage. Cows were also classed into three groups according to individual characteristics. Teat hygiene seems to be the factor that causes the greatest changes in the level of microbial flora on the teat. Whatever the type of practice, rather young cows are associated with lower levels of microbial flora on the teat skin. The microbial community on cow teat skin belonging to 3 groups of farms was evaluated by combining a culture-dependent method and a direct molecular approach. The diversity of this microbial community was large as 79.8% of clones corresponded to unidentified and 66 identified species belonging to most of those commonly found in raw milk. Then teat skin is an interesting source or vector of biodiversity for milk