The use of early subcultures to reduce culturomics handling time

Culturomics, a high scale culture approach based on the multiplication of culture conditions, coupled with extended incubation times proved effective for discovering unknown microorganisms. While significant efforts were made to reduce the number of conditions, preliminary work suggested that strict anaerobic species do not necessarily require prolonged pre-incubation time. This study applies culturomics to faecal samples with one pre-incubation step, following very early and late subcultures. Two stool samples from the same donor were cultured using a single culture condition consisting of pre-incubation in a blood culture bottle containing modified YCFA (Yeast Casitone Fatty Acids Agar), supplemented with blood and rumen, with and without an anaerobic chamber. Subcultures were performed at early (H0 - H24) and late (D2 - D30) stages. A total of 88 bacterial species were isolated from the first stool sample without an anaerobic chamber, including Christensenella and Faecalibacterium species. Subculturing at H0 was the most diverse subculture time, and species considered as fastidious, including Coprococcus comes, Faecalibacterium massiliense and Ruminococcus torques, were subcultured immediately following pre-incubation. We then reduced the less fertile subculture times by reducing their number from 15 to 8 and validated our protocol on a second sample, from which we cultured 103 bacterial species, mostly strict anaerobes. This study demonstrates that strict anaerobes do not require extended pre-incubation times for their growth. Despite limited data from a single stool sample, we propose a rapid and efficient culturomics protocol for anaerobes, using early and late subcultures from one condition, paving the way for high-scale culturomics studies.