Development and validation of a new dynamic in vitro model of the piglet colon (PigutIVM): application to the study of probiotics

Abstract

For ethical, technical, regulatory, and cost reasons, in vitro methods are increasingly used as an alternative to in vivo experimentations. The aim of the present study was to validate, according to in vivo data in living animals, a new in vitro model of the piglet colon, the PigutIVM, under both control conditions and antibiotic disturbance by the widely used colistin. The PigutIVM reproduces the main biotic and abiotic parameters of the piglet colon: temperature, pH, retention time, supply of ileal effluents, complex, and metabolically active microbiota and self-maintained anaerobiosis. Under both control and antibiotic-treated conditions, qPCR analyses showed that the main bacterial populations of piglet gut microbiota were similar in vitro and in vivo, with Pearson correlation coefficient higher than 0.9. During colistin administration, both in piglets and in the in vitro model, a significant decrease in Escherichia coli populations was observed together with changes in microbial composition of subdominant populations. SCFA concentrations were similar in vitro and in vivo and were not modified by colistin. Interestingly, the administration of the probiotic Saccharomyces cerevisiae var. boulardii CNCM I-1079 led in vitro to a decrease in E. coli levels, as previously observed when the antibiotic treatment was applied. This new in vitro model of the piglet colon provides a flexible, reproducible, and cost-effective tool for the screening of drugs or new dietary compounds, such as pre- or probiotics. It will be helpful for researchers, feed producers, or veterinarians when developing innovative non-antibiotic strategies.

Weaning-associated feed deprivation stress causes microbiota disruptions in a novel mucin-containing in vitro model of the piglet colon (MPigut-IVM)

“…Additionally, the MPigut-IVM was able to maintain an archaeal microbiota, composed of Methanobacteriaceae and Methanomethylophilaceae consistently with in vivo data [ 26 ], which was hitherto not observed in any other in vitro model [ 20 , 21 , 24 ]…”

“…We developed a novel in vitro technology to accurately mimic the piglet gut. As opposed to existing models [ 21 , 24 , 47 , 48 ], the MPigut-IVM possesses a unique multiparametric set up by incorporating a mucus environment, an independently established anaerobic redox condition, a fine-tuned and representative piglet feed and a physiologically representative residence time [ 27 ]. Our newly developed MPigut-IVM shows good reproducibility between replicates while maintaining some inter-inocula specificities and displays consistent shifts from a fecal to a colon microbiota such as the increase of the Bacteroidetes phylum [ 26 ].…”

“…Our newly developed MPigut-IVM shows good reproducibility between replicates while maintaining some inter-inocula specificities and displays consistent shifts from a fecal to a colon microbiota such as the increase of the Bacteroidetes phylum [ 26 ]. The augmentation of Bacteroidetes and Proteobacteria relative abundance is commonly observed in in vitro models due to initial quite high oxygen concentration and reactor startup [ 20 , 21 , 48 – 50 ]. We could notice that very few differences were observed in the microbiota composition between technical replicates of control assays (fermentations #1 and 2) and sampling time, compared with the biological replicate (fermentation #3).…”

“…Regarding pH setup, we measured the pH of all the GIT compartments of weaning piglets, and the colonic pH values were all between 6 and 6.5 [ 26 ]. For the setup of the MPigut-IVM we chose to fix the pH to 6, the lower range of our in vivo data, in order to limit the expansion of the Bacteroidetes and Enterobacteriaceae which is most of the time observed in in vitro models during the stabilization period [ 21 , 47 ]. The choice of nutritive medium components has of course also a great impact in modelling in vitro microbiota.…”

“…The constant arrival speed of the nutritive medium inside the bioreactors and the regular withdraw of the fermentation medium ensured a retention time of 19.6 h to mimic the colonic retention time of 4-week old piglets [ 27 ]. A nutritive medium formula containing various sources of carbohydrates, proteins, minerals and vitamins was set up using both the method used by McFarlane et al [ 28 ] , Fleury et al [ 21 ] and on the basis of ingredients generally found in piglet feed at weaning, digestibility indices of ingredients,and the nutrients or growth factors necessary to maintain a viable microbiota in bioreactors (Supplementary Table 1 ). Calculations were based on estimation that piglets of this age would have a feed intake of 500 g per day.…”