The gut microbiota in cystic fibrosis: a complex community unveiled by meta-omics approaches'

In cystic fibrosis (CF) patients the normal community of commensal bacteria in the gut can be affected by intestinal exocrine alfunction, antibiotic usage and swallowing of infected respiratory mucus. However, CFrelated gut dysbiosis has only recently been subjected to detailed investigation. Herein we report on the integrated metaproteomic, metagenomic and metabolomic workflow for monitoring gut microbiota composition. Twenty-four faecal samples were collected from cystic fibrosis young patients (age range 0-6 years) and 6 faecal samples from healthy children. All samples were processed for peptide, DNA and metabolite analysis. 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. In order to evaluate the volatile metabolites a gas-chromatographic (GC)–mass spectrometer (MS)/solid-phase microextraction (SPME) analysis was performed. 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 and GC-MS/SPME were used to identify and quantify fecal metabolites. The application of integrated meta-omics-based approaches to the characterization of the membership and dynamics of the polymicrobial communities colonizing the gut is expected to have important predictive potential in translational medicine, specially in CF-related gut dysbiosis.