Optimization of oligotrophic culturomics for isolation of human gut microbiota

Abstract:

[Objective] To explore the oligotrophic culturomics for the isolation of human gut microbiota. [Methods] Oligotrophic media were obtained by diluting the enrichment medium (blood culture bottle supplemented with sheep blood and rumen fluid), yeast casitone fatty acid (YCFA) agar plate, and YCFA broth. Healthy human fecal samples were cultured with the original medium (0), 5, 10, 20, 30, and 40-fold dilutions of enrichment medium. At different time points (0, 3, 6, 9, 15, 27, and 30 d) during the enrichment, the YCFA agar plate was used to isolate colonies, and then single colonies were picked for further enrichment with YCFA broth. The strains were identified by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry and 16S rRNA gene sequencing. The effects of the above six oligotrophic conditions on the isolation of gut microbiota were compared. Three enrichment media with better isolation effects, i.e., 0-, 10-, and 30-fold dilutions of the enrichment medium, were selected and combined with YCFA agar plate and YCFA broth with the same dilution factors to form nine culture conditions for the optimization of the culture conditions of gut microbiota. [Results] Among the six oligotrophic enrichment media, the 0-, 10-, and 30-fold dilutions of the enrichment medium isolated more species than the other dilutions, and the 10-fold dilution isolated the most species. Twenty-four species were only isolated by the oligotrophic media and not by the original medium. Further optimization showed that the following three combinations isolated the most bacterial species:0-fold diluted enrichment medium+10-fold diluted YCFA plate and broth, 10-fold diluted enrichment medium+0- or 10-fold diluted YCFA plate and broth. As for the three combinations, 20 species were only isolated by the oligotrophic media, and the combination of 10-fold diluted enrichment medium+0-fold diluted YCFA plate and broth isolated the largest number of species. [Conclusion] The oligotrophic culture conditions obtained by diluting the enrichment medium, YCFA agar plate, and YCFA broth can isolate about 40% of species that cannot be isolated by conventional culture conditions, which provide an effective method for isolating more species from the human gut microbiota.

Key words:gut microbiota;oligotrophy;pre-culture;culturomics