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.

Severe acute malnutrition (SAM) is associated with inadequate diet, low levels of plasma antioxidants and gut microbiota alterations. The link between gut redox and microbial alterations, however, remains unexplored. By sequencing the gut microbiomes of 79 children of varying nutritional status from three centers in Senegal and Niger, we found a dramatic depletion of obligate anaerobes in malnutrition. This was confirmed in an individual patient data meta-analysis including 107 cases and 77 controls from 5 different African and Asian countries. Specifically, several species of the Bacteroidaceae, Eubacteriaceae, Lachnospiraceae and Ruminococceae families were consistently depleted while Enterococcus faecalis, Escherichia coli and Staphylococcus aureus were consistently enriched. Further analyses on our samples revealed increased fecal redox potential, decreased total bacterial number and dramatic Methanobrevibacter smithii depletion. Indeed, M. smithii was detected in more than half of the controls but in none of the cases. No causality was demonstrated but, based on our results, we propose a unifying theory linking microbiota specificity, lacking anaerobes and archaea, to low antioxidant nutrients, and lower food conversion.

Anaerosalibacter massiliensis sp. nov. strain ND1(T) (= CSUR P762 = DSM 27308) is the type strain of A. massiliensis sp. nov., a new species within the genus Anaerosalibacter. This strain, the genome of which is described here, was isolated from the faecal flora of a 49-year-old healthy Brazilian man. Anaerosalibacter massiliensis is a Gram-positive, obligate anaerobic rod and member of the family Clostridiaceae. With the complete genome sequence and annotation, we describe here the features of this organism. The 3 197 911 bp long genome (one chromosome but no plasmid) contains 3271 protein-coding and 62 RNA genes, including six rRNA genes.

In the mid-19th century, the dichotomy between aerobic and anaerobic bacteria was introduced. Nevertheless, the aerobic growth of strictly anaerobic bacterial species such as Ruminococcus gnavus and Fusobacterium necrophorum, in a culture medium containing antioxidants, was recently demonstrated. We tested aerobically the culture of 623 bacterial strains from 276 bacterial species including 82 strictly anaerobic, 154 facultative anaerobic, 31 aerobic and nine microaerophilic bacterial species as well as ten fungi. The basic culture medium was based on Schaedler agar supplemented with 1 g/L ascorbic acid and 0.1 g/L glutathione (R-medium). We successively optimized this media, adding 0.4 g/L uric acid, using separate autoclaving of the component, or adding haemin 0.1 g/L or α-ketoglutarate 2 g/L. In the basic medium, 237 bacterial species and ten fungal species grew but with no growth of 36 bacterial species, including 22 strict anaerobes. Adding uric acid allowed the growth of 14 further species including eight strict anaerobes, while separate autoclaving allowed the growth of all tested bacterial strains. To extend its potential use for fastidious bacteria, we added haemin for Haemophilus influenzae, Haemophilus parainfluenzae and Eikenella corrodens and α-ketoglutarate for Legionella pneumophila. This medium allowed the growth of all tested strains with the exception of Mycobacterium tuberculosis and Mycobacterium bovis. Testing primoculture and more fastidious species will constitute the main work to be done, but R-medium coupled with a rapid identification method (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry) will facilitate the anaerobic culture in clinical microbiology laboratories.

Molecular approaches have long led to the assumption that the human gut microbiota is dominated by uncultivable bacteria. The recent advent of large-scale culturing methods, and in particular that of culturomics have demonstrated that these prokaryotes can in fact be cultured. This is increasing in a dramatic manner the repertoire of commensal microbes inhabiting the human gut. Following eight years of culturomics approach applied on more than 900 samples, we propose herein a remake of the pioneering study applying a dual approach including culturomics and metagenomics on a cohort of 8 healthy specimen. Here we show that culturomics enable a 20% higher richness when compared to molecular approaches by culturing 1 archaeal species and 494 bacterial species of which 19 were new taxa. Species discovered as a part of previous culturomics studies represent 30% of the cultivated isolates, while sequences derived from these new taxa enabled to increase by 22% the bacterial richness retrieved by metagenomics. Overall, 67% of the total reads generated were covered by cultured isolates, significantly reducing the hidden content of sequencing methods compared to the pioneering study. By redefining culture conditions to recover microbes previously considered fastidious, there are greater opportunities than ever to eradicate metagenomics dark matter.