pig gut metagenome Metagenome

We used plant-diet derived digesta inocula prepared from jejunal and colonic digesta of weaned piglets to study the mucosal response pre- and postweaning towards the microbial and metabolite composition in the inocula. In order to do so the jejunal and colonic inocula were perfused into intestinal loops in a so called intestinal loop perfusion assay. To characterize potential differences in the bacterial metabolic activity during the 2-hour lasting perfusion, we applied fluorescent tracking with click chemistry based on bioorthogonal noncanonical amino acid tagging (BONCAT) followed by 16S rRNA gene amplicon sequencing. For this, we incubated the jejunal and colonic digesta for 90 min with or without L-azidohomoalanine (L-AHA) which enables click-chemistry by an alkyne-azide reaction and is an alternative methionine source, and the samples were incubated at body temperature for 0 and 90 min. The L-AHA-positive bacteria (C5 and Syto 9 positive) were sorted from the L-AHA-negative bacteria (only Syto 9 positive) using fluorescence activated cell sorting (FACS). Thereafter, DNA was extracted from the incubated POM inocula as described above. For taxonomic abundances, the V3-V4 hypervariable regions of bacterial 16S rRNA gene was amplified using a 250 bp read length paired-end protocol (Microsynth, Balgach, Switzerland) on an Illumina MiSeq sequencing v2 platform, including the 16S rRNA PCRs, library preparation and sequencing.