Running after ghosts: are dead bacteria the dark matter of the human gut microbiota?

The human gut microbiota has been explored by a wide range of culture-dependent and culture-independent methods, revealing that many microbes remain uncharacterized and uncultured. In this work, we aimed to confirm the hypothesis that some of the species present in the human gut microbiota remain uncultured not because of culture limitations, but because all members of such species are dead before reaching the end of the gastro-intestinal tract. We evaluate this phenomenon by studying the microbial viability and culturability of the human gut microbiota from the fresh fecal materials of eight healthy adults. For the first time, we applied fluorescence-activated cell sorting (FACS) combined with 16S metagenomics analysis and microbial culturomics. We identified a total of 1,020 bacterial OTUs and 495 bacterial isolates through metagenomics and culturomics, respectively. Among the FACS metagenomics results, only 735 bacterial OTUs were alive, comprising on average 42% of known species and 87% of relative abundance per individual. The remaining uncultured bacteria were rare, dead, or injured. Our strategy allowed us to shed light on the dark matter of the human gut microbiota and revealed that both metagenomics and culturomics approaches are needed for greater insight into the diversity and richness of bacteria in the human gut microbiota. Further work on culture is needed to enhance the repertoire of cultured gut bacteria by targeting low abundance bacteria and optimizing anaerobic sample conditioning and processing to preserve the viability of bacteria.

Automated summary

This study investigated whether some microbial species in the human gut microbiota remain uncultured because they are dead before reaching the end of the gastrointestinal tract. By combining flow cytometry and 16S metagenomics analysis with microbial culturomics, it was revealed that only a portion of bacteria were alive, while the rest of the uncultured bacteria were rare, dead, or injured. This highlights the importance of both metagenomics and culturomics approaches for a comprehensive understanding of the diversity in the human gut microbiota.