Succession of early microbial consortia in the developing infant intestinal microbiota unveiled by meta-omics approaches

Immediately after birth, the intestinal microbiota development is orchestrated by several variability determinants, including feeding modality. Herein we report on the integrated metaproteomic, metagenomic and metabolomic workflow for monitoring gut microbiota development. Ninety-six faecal samples were collected at birth, 2, 3, 7, 15, 30 days from 23 at-term caesarean section delivered neonates, of which 17/23 breast- (BF) and 6/23 formula-fed (FF), respectively. Twelve samples from one BF and one FF neonates were processed for peptide, DNA and metabolite analysis throughout the time-course. Protein content was analyzed with a shotgun metaproteomic approach by a nano-scale LC-MS2 set-up (Bruker amaZon-ETD) and filtered for operational taxon units (OTUs) assignment. OTUs were corroborated by hybridization of the small subunit ribosomal RNA (SSU rRNA) gene variable regions against the human intestinal tract microarray platform HITChip. 1H-NMR spectra were acquired on a Varian 400 MHz Mercury-plus NMR . Pre-analytical steps of the metaproteomics workflow were devised to improve hit coverage, while the OTUs repository was guaranteed by metagenomics entries. 1H-NMR was used to identify and quantify fecal metabolites. The amount of pyruvic, citric and acetic acids was found to increase in the FF neonate, while others such as adenosine monophosphate increased in the BF neonate. Several amino acids, including valine, tyrosine, glycine, leucine and alanine, showed an initial decrease, followed by an increase in both neonates. Early life colonization may impact later health and hence the application of parallel and integrated meta-omics-based approaches to neonatal samples is expected to have important predictive potential in translational medicine.