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

## TL;DR

A high-throughput method successfully isolates diverse Bifidobacterium strains from human gut samples, enabling detailed study of their genetic and functional diversity.

## Contribution

A novel high-throughput single-cell dispensing workflow is introduced for efficient isolation of diverse Bifidobacterium strains from fecal samples.

## Key findings

- The workflow achieved high single-cell dispensing frequency (>88%) and preserved species diversity.
- 622 viable isolates were obtained, with >98% identified as Bifidobacterium species.
- 96 isolates sequenced revealed six Bifidobacterium species, with >66% being novel to public databases.

## Abstract

Bifidobacterium represents a diverse genus of commensal gut bacteria with key roles in human health, from metabolizing indigestible fibers to protecting against pathogens. While metagenomic studies have highlighted significant strain diversity for Bifidobacterium species within individuals, their systematic isolation and phenotypic characterization can be hampered by the significant effort and biases inherent in traditional culturomics. Here, we explored the utility of a high-throughput single-cell dispensing system (B.SIGHT)-based workflow for accelerating the process of isolating diverse Bifidobacterium strains from fecal samples. Systematic assessment of this workflow revealed a high single-cell dispensing frequency (>88%) and the ability to preserve species diversity when a pool of Bifidobacterium strains was dispensed. Culturing-related factors including the use of an effective selection medium, such as the Bifidus Selective Medium supplemented with mupirocin, and the length of pre-dispensing incubation were found to be critical in determining isolation success. Leveraging this workflow, we obtained a total of 622 viable isolates from five Singaporean fecal samples, of which >98% were found to be from Bifidobacterium species. Whole-genome sequencing of 96 isolates identified six different Bifidobacterium species with both inter- and intra-subject strain and lineage diversity, and the majority (>66%) were novel relative to large public genomic databases. Our findings highlight the ability of this high-throughput culturomics workflow to accelerate the recovery of diverse and novel Bifidobacterium strains, enabling further interrogation of their functional characteristics and advancing our understanding of important bacterial species in the gut microbiome.

The field of high-throughput microbial culturomics is still in its early stages. Enhancing our ability to isolate and phenotypically test bacterial strains from complex communities is crucial for advancing microbiome research and healthcare development. Given the time and cost inefficiencies of traditional culturing methods, a more efficient, high-throughput approach to obtain isolates is needed. In the present study, we assessed a single-cell dispensing platform and developed a workflow to isolate diverse Bifidobacterium strains from fecal samples. We demonstrated here the capability of this novel technology to efficiently obtain hundreds of isolates of a targeted group, covering both species and strain diversities. This generalizable and scalable method can potentially allow for the high-throughput recovery of microbes from other taxonomic groups, providing a fundamental step in improving the culturomics framework to complement metagenomic approaches and enable isolate-level functional studies of important microbes.