High-throughput single-cell isolation of Bifidobacterium strains from the gut microbiome

While metagenomic studies can highlight strain-level diversity within microbial communities, the diversity obtained is often incomplete. Moreover, their utility for phenotypic characterizations remains hampered without the subjacent, systematic isolation procedures required with traditional culturomics. In this work, we examined the capabilities of a commercially available high-throughput single-cell dispensing solution to selectively target and isolate diverse strains of a genus of interest, Bifidobacterium , from fecal samples. The general performance of the single-cell dispenser was first assessed, revealing a low doublet frequency of 11.5% and an ability to preserve global genus diversity when a mixed culture of Bifidobacterium was dispensed. Culturing-related factors including the use of an effective selection medium, such as the Bifidus Selective Medium supplemented with mupirocin (BSM-MUP), and the length of incubation were found to be critical in determining isolation success. Leveraging these results, we obtained a total of 622 viable isolates from five Singaporean fecal samples, among which ∼98.7% were bifidobacteria. Whole-genome sequencing of 96 isolates revealed six different Bifidobacterium species with both inter- and intra-subject lineage diversity, and the majority of the assemblies were not previously captured using metagenomic sequencing. Our findings validate the ability of high-throughput culturomics to recover diverse, novel bacterial strains and open up the possibility to robustly interrogate their functional characteristics, advancing our understanding of important microbiomes.