Culture of previously uncultured members of the human gut microbiota by culturomics

Abstract

Metagenomics revolutionized the understanding of the relations among the human microbiome, health and diseases, but generated a countless number of sequences that have not been assigned to a known microorganism¹. The pure culture of prokaryotes, neglected in recent decades, remains essential to elucidating the role of these organisms². We recently introduced microbial culturomics, a culturing approach that uses multiple culture conditions and matrix-assisted laser desorption/ionization-time of flight and 16S rRNA for identification². Here, we have selected the best culture conditions to increase the number of studied samples and have applied new protocols (fresh-sample inoculation; detection of microcolonies and specific cultures of Proteobacteria and microaerophilic and halophilic prokaryotes) to address the weaknesses of the previous studies^3-5. We identified 1,057 prokaryotic species, thereby adding 531 species to the human gut repertoire: 146 bacteria known in humans but not in the gut, 187 bacteria and 1 archaea not previously isolated in humans, and 197 potentially new species. Genome sequencing was performed on the new species. By comparing the results of the metagenomic and culturomic analyses, we show that the use of culturomics allows the culture of organisms corresponding to sequences previously not assigned. Altogether, culturomics doubles the number of species isolated at least once from the human gut.

Figure 1. Number of different bacteria and archaea isolated during the culturomics studies.

Columns A and B represent the results from previously published studies, and columns C to K the different projects described herein. The bacterial species are represented in five categories: NS, new species; NH, prokaryotes first isolated in humans; H, prokaryotes already known in humans but never isolated from the human gut; H (GUT), prokaryotes known in the human gut but newly isolated by culturomics; and prokaryotes isolated by other laboratories but not by culturomics.

Figure 2

Summary of the culturomics work that has extended the gut repertoire and filled some of the gaps in metagenomics.

Figure 3. Phylogenetic tree of the 247 new prokaryote species isolated by culturomics.

Bacterial species from Firmicutes are highlighted in red, Actinobacteria (light green), Proteobacteria (blue), Bacteroidetes (purple), Synergistetes (green), Fusobacteria (dark green) and Archaea (grey), respectively. The sequences of 16 prokaryotic species belonging to six phyla previously known from the human gut and more frequently isolated by culture in human gut are highlighted in bold and by an asterisk.