A Guide for Ex Vivo Handling and storage of stool samples Intended for Fecal Microbiota transplantation

International audienceOwing to the growing recognition of the gut microbiota as a main partner of human health, we are expecting that the number of indications for fecal microbiota transplantation (FMt) will increase. Thus, there is an urgent need for standardization of the entire process of fecal transplant production. This study provides a complete standardized procedure to prepare and store live and ready-to-use transplants that meet the standard requirements of good practices to applied use in pharmaceutical industry. We show that, if time before transformation to transplants would exceed 24 hours, fresh samples should not be exposed to temperatures above 20 °C, and refrigeration at 4 °C can be a safe solution. Oxygen-free atmosphere was not necessary and simply removing air above collected samples was sufficient to preserve viability. Transplants prepared in maltodextrin-trehalose solutions, stored in a-80 °C standard freezer and then rapidly thawed at 37 °C, retained the best revivification potential as proven by 16S rRNA profiles, metabolomic fingerprints, and flow cytometry assays over a 3-month observation period. Maltodextrin-trehalose containing cryoprotectants were also efficient in preserving viability of lyophilized transplants, either in their crude or purified form, an option that can be attractive for fecal transplant biobanking and oral formulation

Endoglucanase activity and relative expression of glycoside hydrolase genes of Fibrobacter succinogenes S85 grown on different substrates

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Fiber-Degrading Systems of Different Strains of the Genus Fibrobacter

The S85 type strain of Fibrobacter succinogenes, a major ruminal fibrolytic species, was isolated 49 years ago from a bovine rumen and has been used since then as a model for extensive studies. To assess the validity of this model, we compared the cellulase- and xylanase-degrading activities of several other F. succinogenes strains originating from different ruminants, including recently isolated strains, and looked for the presence of 10 glycoside hydrolase genes previously identified in S85. The NR9 F. intestinalis type strain, representative of the second species of the genus, was also included in this study. DNA-DNA hybridization and 16S rRNA gene sequencing first classified the strains and provided the phylogenetic positions of isolates of both species. Cellulase and xylanase activity analyses revealed similar activity profiles for all F. succinogenes strains. However, the F(E) strain, phylogenetically close to S85, presented a poor xylanolytic system and weak specific activities. Furthermore, the HM2 strain, genetically distant from the other F. succinogenes isolates, displayed a larger cellulolytic profile on zymograms and higher cellulolytic specific activity. F. intestinalis NR9 presented a higher cellulolytic specific activity and a stronger extracellular xylanolytic activity. Almost all glycoside hydrolase genes studied were found in the F. succinogenes isolates by PCR, except in the HM2 strain, and few of them were detected in F. intestinalis NR9. As expected, the fibrolytic genes of strains of the genus Fibrobacter as well as the cellulase and xylanase activities are better conserved in closely related phylogenetic isolates

Gene sequence analysis and properties of EGC, a family E (9) endoglucanase from Fibrobacter succinogenes BL2

International audienceThe endoglucanase gene (endC) of Fibrobacter succinogenes BL2 encodes a protein of 620 amino acids (EGC) that shows similarity with family E1 cellulases, and particularly with EGB from F. succinogenes S85. Alignment of the amino acid sequence of family E1 cellulases revealed that EGC is composed of a N-terminal domain and a large catalytic domain of 453 residues containing an extension of 60 residues at its C-terminal part which is not present in other family E1 enzymes. EGC shows the same substrate specificity as EGB, and is also inhibited by EDTA. However, its optimal pH (7.0) and temperature (37 degrees C) for activity are different

Quantification by real-time PCR of cellulolytic bacteria in the rumen of sheep after supplementation of a forage diet with readily fermentable carbohydrates: effect of a yeast additive

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Biochemical characterization of MI-ENG1, a family 5 endoglucanase secreted by the root-knot nematode Meloidogyne incognita

International audienceA beta-1,4-endoglucanase named MI-ENG1, homologous to the family 5 glycoside hydrolases, was previously isolated from the plant parasitic root-knot nematode Meloidogyne incognita. We describe here the detection of the enzyme in the nematode homogenate and secretion and its complete biochemical characterization. This study is the first comparison of the enzymatic properties of an animal glycoside hydrolase with plant and microbial enzymes. MI-ENG1 shares many enzymatic properties with known endoglucanases from plants, free-living or rumen-associated microorganisms and phytopathogens. In spite of the presence of a cellulose-binding domain at the C-terminus, the ability of MI-ENG1 to bind cellulose could not be demonstrated, whatever the experimental conditions used. The biochemical characterization of the enzyme is a first step towards the understanding of the molecular events taking place during the plant-nematode interaction

Mise au point d'un système de pesée pour rongeurs élevés en isolateur stérile

National audienceCet article décrit un dispositif expérimental conçu pour peser de petits animaux en isolateurs stériles sans sortir les animaux de leur environnement et sans introduire le matériel de pesée pour chaque mesure. Ce dispositif a été conçu et réalisé par les techniciens animaliers de l’installation expérimentale de l’unité de Microbiologie UR454 du Centre INRA de Clermont-Ferrand/Theix. Il est constitué d’une partie fixe (balance) posée à l’extérieur de l’isolateur et d’une partie mobile (plateau de pesée) disposée dans l’isolateur

Comparative Study of Bacterial Groups within the Human Cecal and Fecal Microbiota

International audienceABSTRACT The composition of the human cecal microbiota is poorly known because of sampling difficulties. Samples of cecal fluid from eight subjects were collected via an intestinal tube. Feces were also collected. Total anaerobes, facultative anaerobes, bifidobacteria, and Bacteroides were enumerated by culture methods, and the predominant phylogenetic groups were quantified by molecular hybridization using a set of six rRNA-targeted probes. The numbers of strict anaerobes, bifidobacteria, Bacteroides , and members of the Clostridium coccoides group and Clostridium leptum subgroup were lower in the cecum. Facultative anaerobes represented 25% of total bacteria in the cecum versus 1% in the feces

Development of a RT-qPCR method for the quantification of Fibrobacter succinogenes S85 glycoside hydrolase transcripts in the rumen content of gnotobiotic and conventional sheep

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Characterisation of endoglucanases EGB and EGC from Fibrobacter succinogenes

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