Active and secreted IgA-coated bacterial fractions from the human gut

State of the art. During the last decade massive sequencing methods have elucidated the diversity of human-associated microbiota from several body parts. While it would seem clear that microbes perform diverse activities and vary in distribution depending on their operational zone, activity and mechanisms underlying their interactions with the human immune system still pose a research challenge. It has been observed that secreted immunoglobulin coated (S-IgA-coated) bacteria, despite being opsonised, can belong to the group of “indigenous microbes” which would not induce strong immune responses and that inflammatory processes can be activated not only in the case of pathogen invasion but also in a dysbiosis scenario. Methods. In this work, we used a single-cell based approach to describe the taxonomic distribution of faecal microbiota. Both active bacteria and S-IgA-coated bacterial fractions from six healthy adult volunteers were studied. Cells were stained with pyronin-Y or with anti-human-S-IgA secondary antibodies labelled with fluorescein isothiocyanate (FITC). We then used flow cytometry and cell sorting to obtain 1) active bacteria as evidenced by their RNA content and 2) S-IgA-coated bacterial fractions. We studied taxonomic composition using 16S rDNA pyrosequencing. We also obtained microbiota taxonomic profiles of samples from raw faecal material, which is the most common approach used to study gastrointestinal microbiota, and subsequently compared the results with active and S-IgA-coated fractions obtained by flow cytometry. Results. Active and S-IgA-coated bacterial fractions were characterized by a higher diversity than that observed in faecal suspensions. We revealed the importance of the long-tail effect observed in family distribution where under-represented bacteria can cover up to 20 % of total diversity. Moreover, these under-represented taxa contribute an important fraction of the shared diversity, defining a structured microbiota network. We revealed that Firmicutes is not only the most represented phylum, but it is also the one hosting the majority of sequences that were not assigned at the genus level with the confidence threshold used. Finally, many under-represented taxa, for example members of Sphingomonadaceae, were found to be part of the active or S-IgA-coated core microbiota.