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International audience

International audience

International audience

International audience

International audience

Session 3: New approaches to study the gut microbiome : Communications P 127; International audience; Irritable bowel syndrome (IBS) is associated with alterations of gut microbiota, highlighting its importance in the disease onset or evolution. Animal models are commonly used in preclinical phases to study therapeutic strategies for IBS, but this approach remains limited by main differences between human and animal digestive physiology, including gut microbiota. In vitro digestion models can offer a great alternative to in vivo assays to perform mechanistic studies. However, there is no relevant in vitro system reproducing the colonic environment of IBS patients. In this context, an in-depth literature review was performed to adapt the mucosal Artificial Colon (M-ARCOL) model to the specific conditions found in diarrheic IBS (IBS-D) patients. This in vitro model reproduces both the physicochemical (pH, temperature, transit time, nutrient availability and anaerobiosis) and microbial (luminal and mucus-associated microbiota) parameters of the human colonic environment, though inoculation with faecal samples. One stool was used to inoculate two bioreactors ran in parallel, one set-up with healthy conditions, the other with IBS-D parameters (n=4). Samples were regularly collected to evaluate gut microbiota composition (qPCR and 16S sequencing) and metabolic activity (gas and short chain fatty acid production). In IBS-D condition, a tendency to produce less SCFAs and energy was shown. A decrease in Archaea and Ruminococcaceae, together with an increase in Akkermansia, Veillonellaceae and Proteobacteria was observed in accordance with in vivo data. This study brings first evidence that reproducing physicochemical parameters of IBS-D colon was sufficient to induce shift in microbiota structure, close to the in vivo situation. In a next step, the development of the dysbiotic model will be performed by inoculating bioreactors with stool samples from IBS-D patients. Once fully validated, this in vitro model may help to test strategies aiming to restore gut microbiota eubiosis such as pre- or probiotics supplementation or faecal microbiota transplantation.

Ação n.º: TC-14/2017 UIDB/50006/2020 The commensal bacteria Escherichia coli causes several intestinal and extra-intestinal diseases, since it has virulence factors that interfere in important cellular processes. These bacteria also have a great capacity to spread the resistance genes, sometimes to phylogenetically distant bacteria, which poses an additional threat to public health worldwide. Here, we aimed to use the analytical potential of MALDI-TOF mass spectrometry (MS) to characterize E. coli isolates and identify proteins associated closely with antibiotic resistance. Thirty strains of extended-spectrum beta-lactamase producing E. coli were sampled from various animals. The phenotypes of antibiotic resistance were determined according to Clinical and Laboratory Standards Institute (CLSI) methods, and they showed that all bacterial isolates were multi-resistant to trimethoprim-sulfamethoxazole, tetracycline, and ampicillin. To identify peptides characteristic of resistance to particular antibiotics, each strain was grown in the presence or absence of the different antibiotics, and then proteins were extracted from the cells. The protein fingerprints of the samples were determined by MALDI-TOF MS in linear mode over a mass range of 2 to 20 kDa. The spectra obtained were compared by using the ClinProTools bioinformatics software, using three machine learning classification algorithms. A putative species biomarker was also detected at a peak m/z of 4528.00. publishersversion published

Additional file 1: Fig. S1. [Global ETEC survival percentages in the cumulated ileal effluents of the TIM-1]. Fig. S2. [Live/Dead flow cytometry for accurate determination of ETEC membrane physiology in the TIM-1 gastric and ileal effluents]. Fig. S3. [Spearman correlation between virulence gene expression, LT toxin production and/or ETEC survival in the TIM-1 and M-SHIME]. Fig. S4. [Phylum and species levels microbial community composition of the luminal gut regions]. Fig. S5. [Phylum and species levels microbial community composition of the mucosal gut regions]. Fig. S6. [Gut microbes concentration in the ileum and ascending colon lumen determined by total flora 16S rRNA gene quantification]. Fig. S7. [Simpson alpha diversity index over time for the six donors according to the luminal (L) and mucosal (M) gut regions]. Fig. S8. [db-RDA triplots showing the relationship of A) gut regions, B) donors and C) period pre- vs post-infection as explanatory variables to the microbial community structure at genus level]. Fig. S9. [db-RDA triplots showing the effect of ETEC pre- and post-infection on distinct microbial genera according to the gut regions]. Fig. S10. [SCFA concentrations (mM) in the ileum (ILE) and ascending colon (ASC) compartments of the M-SHIME]. Fig. S11. [Spearman correlations between the main SCFA produced in the ascending colon and ETEC survival, LT toxin production, and expression of virulence genes encoding for the enterotoxins]. Fig. S12. [High reproducibility in SCFA concentrations and microbiota composition between replicates from a same donor in a separate SHIME experiment]. Table S1. [ETEC intracellular pH (pHi) in the TIM-1 system]. Table S2. [ETEC membrane potential in the TIM-1 system]. Table S3. [Log2 fold changes in virulence genes expression in the TIM-1]. Table S4. [Log2 fold changes in virulence genes expression in the M-SHIME]. Table S5. [Parameters of the TIM-1 and M-SHIME systems]. Table S6. [General characteristic of the fecal donors involved in the M-SHIME experiments]. Table S7. [Static in vitro gastro-jejunal digestion procedure]. Table S8. [ETEC primers used in the study]. Table S9. [RDP Seqmatch and NCBI BLAST results for the most abundant species and/or species of interest in the M-SHIME].

Background: The availability of fresh stool samples is a prerequisite in most gut microbiota functional studies. Objective: Strategies for amplification and long-term gut microbiota preservation from fecal samples would favor sample sharing, help comparisons and reproducibility over time and between laboratories, and improve the safety and ethical issues surrounding fecal microbiota transplantations. Design: Taking advantage of in vitro gut-simulating systems, we amplified the microbial repertoire of a fresh fecal sample and assessed the viability and resuscitation of microbes after preservation with some common intracellular and extracellular acting cryoprotective agents (CPAs), alone and in different combinations. Preservation efficiencies were determined after 3 and 6 months and compared with the fresh initial microbiota diversity and metabolic activity, using the chemostat-based Environmental Control System for Intestinal Microbiota (ECSIM) in vitro model of the gut environment. Microbial populations were tested for fermentation gas, short-chain fatty acids, and composition of amplified and resuscitated microbiota, encompassing methanogenic archaea. Results: Amplification of the microbial repertoire from a fresh fecal sample was achieved with high fidelity. Dimethylsulfoxide, alone or mixed with other CPAs, showed the best efficiency for functional preservation, and the duration of preservation had little effect. Conclusions: The amplification and resuscitation of fecal microbiota can be performed using specialized in vitro gut models. Correct amplification of the initial microbes should ease the sharing of clinical samples and improve the safety of fecal microbiota transplantation. Abbreviations: CDI, Clostridium difficile infection; CPA, cryoprotective agent; D, DMSO, dimethylsulfoxide; FMT, fecal microbiota transplantation; G, glycerol; IBD, inflammatory bowel disease; P, PEG-4000, polyethylene glycol 4000 g.mol(-1); SCFA, short-chain fatty acid; SNR, signal-to-noise ratio.

National audience; Introduction et but de l’étude : La digestion et l’absorption des protéines de lait solubles dites protéines "rapides", diffèrent de celle des caséines micellaires dites protéines "lentes" (Boirie et al, 1997). Contrairement aux protéines solubles, les caséines coagulent dans l’estomac à pH acide, entrainant un ralentissement de leur vidange gastrique et par conséquent, une libération prolongée des acides aminés dans le sang après digestion. Cependant, peu d’information existe sur le lien structure/digestibilité des protéines, en particulier pour les caséines. Aussi, nous avons étudié in vitro l’impact de la dénaturation des caséines micellaires (CM) en caséinates de sodium (CNa) et de calcium (CCa) sur la dégradation de ces protéines au cours de la digestion. Matériel et méthodes : Les trois protéines (CM, CNa, CCa) ont été digérées dans le système TIM, outil dynamique quadri-compartimenté reproduisant les principaux paramètres mécaniques, physico-chimiques et spatio-temporels de la digestion humaine (Minekus et al, 1995). Les produits (poudres réhydratées à 5%) ont été digérés seuls (n=3) pour faciliter le suivi de leur dégradation, selon un protocole simulant la digestion d’un repas (transit lent) chez l’adulte sain. Un suivi des matières azotés totales libérées, un bilan azoté et un suivi des profils peptidiques (poids moléculaire) ont été réalisés sur les 5h de digestion. Résultats et Analyse statistique : Dans l’estomac, les caséines précipitent rapidement, mais en formant des coagula différents, plus ou moins compacts, qui provoquent un ralentissement global de leur vidange gastrique.La vidange gastrique est significativement plus rapide (P < 0.01) avec les CNa, qu’avec les CM et les CCa. Le profil peptidique des fractions liquides analysées à plusieurs temps est également différent pour les trois produits. Pour CM, la part de macromolécules (>10kDa) reste importante au début de digestion (61% à 50 min) et diminue dans le temps (13% à 5h). Pour CNa, le phénomène est inverse (fraction >10kDa = 11% à 50 min et 38% à 5h) et concorde avec la solubilisation apparente du coagulum.Dans l’intestin grêle, la digestibilité des 3 protéines est très élevée (> 90%). L’absorption se situe majoritairement dans le jéjunum (74 à 84%). Le profil peptidique est similaire pour toutes les protéines aux différents temps d’analyse. Conclusion : Même si les trois formes peuvent être définies comme des protéines lentes selon le concept de Boirie, elles présentent un comportement différent dans l’estomac qui engendre une vidange plus rapide de CNa par rapport à CM et CCa. Une étude rhéologique permettrait de mieux caractériser les phénomènes observés. Le changement de structure modifie le processus de digestion des protéines et donc le métabolisme protéique

Partie IV. Modéliser le devenir digestif de l'aliment21. Les modèles in vitro; Les modèles in vitro

The diversity of methanogenic Archaea from the gut of 6 humans was investigated by targeting mcrA, a molecular metabolic marker of methanogenesis. Three operational taxonomic units (OTUs) were recovered from about 400 clones analyzed, two of which were attributed to the expected Methanobacteriales Methanobrevibacter smithii (4 volunteers) and Methanosphaera stadtmanae (1 volunteer). The third OTU (1 volunteer) corresponded to a distant phylotype that does not cluster with any of the five methanogenic orders. This result, also supported by 16S archaeal sequences retrieved from the same volunteer, strongly suggests there may be a sixth order and hence potential underestimation of the role of methanogens in gut physiology.