Culturomics is a high-throughput culture approach that has dramatically contributed to the recent renewal of culture. While metagenomics enabled substantial advances in exploring the microbiota, culturomics significantly expanded our knowledge regarding the bacterial gut repertoire through the discovery and the description of hundreds of new taxa. While this approach relies on the variation of culture conditions and media, we have tested so far more than 300 conditions since the beginning of culturomics studies. In this context, we aimed herein to identify the most profitable conditions for optimizing culturomics approach. For this purpose, we have analysed a set of 58 culturomics conditions that were previously applied to 8 faecal specimens, enabling the isolation of 497 bacterial species. As a result, we were able to reduce the number of conditions used to isolate these 497 of more than a half (i.e. to 25 culture conditions). We have also established a list of the 16 conditions that allowed to capture 98% of the total number of species previously isolated. These data constitute a methodological starting point for culture-based microbiota studies by improving the culturomics workflow without any loss of captured bacterial diversity.
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.
Human urine was considered sterile for a long time. However, 416 species have been previously cultured, including only 40 anaerobic species. Here, we used culturomics, particularly those targeting anaerobes, to better understand the urinary microbiota. By testing 435 urine samples, we isolated 450 different bacterial species, including 256 never described in urine of which 18 were new species. Among the bacterial species identified, 161 were anaerobes (35%). This study increased the known urine repertoire by 39%. Among the 672 bacterial species isolated now at least once from urine microbiota, 431 (64.1%) were previously isolated from gut microbiota, while only 213 (31.7%) were previously isolated from vagina. These results suggest that many members of the microbiota in the urinary tract are in fact derived from the gut, and a paradigm shift is thus needed in our understanding.
The candidate phyla radiation (CPR) has been described as an obligatory group of ultrasmall bacteria associated with host bacteria. They phylogenetically represent a subdivision of bacteria distinct from other living organisms. Using polyphasic approaches, we screened human faecal samples for the detection of Saccharibacteria. The new sequences obtained by sequencing were compared to the complete CPR genomes available to date. Then, we attempted a co-culture of CPR-bacteria and non-CPR bacteria from human faecal samples. We finally aimed to evaluate the prevalence and distribution of these Saccharibacteria sequences in human sources in 16S amplicon datasets. We were able to reconstitute two high-quality Saccharibacteria genomes named Minimicrobia massiliensis and Minimicrobia timonensis. We have established, for the first time in human digestive samples, the coculture of Candidatus Saccharibacteria with two different bacterial hosts. Finally, we showed that 12.8% (610/4,756) of samples sequenced in our laboratory were positive for operational taxonomic units (OTUs) assigned to M.massiliensis. and significantly enriched in human respiratory and oral microbiota. Here, we reported the first genomes and coculture of Saccharibacteria from human gut specimens. This study opens a new field, particularly in the study of the involvement of CPR in the human intestinal microbiota.
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