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Bifidobacteria represent a dominant constituent of human gut microbiomes during infancy, influencing nutrition, immune development, and resistance to infection. Despite interest in bifidobacteria as a live biotic therapy, our understanding of colonization, host-microbe interactions, and the health-promoting effects of bifidobacteria is limited. To address these major knowledge gaps, we used a large-scale genetic approach to create a mutant fitness compendium in Bifidobacterium breve. First, we generated a high-density randomly barcoded transposon insertion pool and used it to determine fitness requirements during colonization of germ-free mice and chickens with multiple diets and in response to hundreds of in vitro perturbations. Second, to enable mechanistic investigation, we constructed an ordered collection of insertion strains covering 1,462 genes. We leveraged these tools to reveal community- and diet-specific requirements for colonization and to connect the production of immunomodulatory molecules to growth benefits. These resources will catalyze future investigations of this important beneficial microbe. 

Conventional cuvette-based and microfluidics-based electroporation approaches for bacterial gene delivery have distinct advantages, but they are typically limited to relatively small sample volumes, reducing their utility for applications requiring high throughput such as the generation of mutant libraries. Here, we present a scalable, large-scale bacterial gene delivery approach enabled by a disposable, user-friendly microfluidic electroporation device requiring minimal device fabrication and straightforward operation. We demonstrate that the proposed device can outperform conventional cuvettes in a range of situations, including across Escherichia coli strains with a range of electroporation efficiencies, and we use its large-volume bacterial electroporation capability to generate a library of transposon mutants in the anaerobic gut commensal Bifidobacterium longum . 

Abstract The spatiotemporal structure of the human microbiome^1,2, proteome³and metabolome^4,5reflects and determines regional intestinal physiology and may have implications for disease⁶. Yet, little is known about the distribution of microorganisms, their environment and their biochemical activity in the gut because of reliance on stool samples and limited access to only some regions of the gut using endoscopy in fasting or sedated individuals⁷. To address these deficiencies, we developed an ingestible device that collects samples from multiple regions of the human intestinal tract during normal digestion. Collection of 240 intestinal samples from 15 healthy individuals using the device and subsequent multi-omics analyses identified significant differences between bacteria, phages, host proteins and metabolites in the intestines versus stool. Certain microbial taxa were differentially enriched and prophage induction was more prevalent in the intestines than in stool. The host proteome and bile acid profiles varied along the intestines and were highly distinct from those of stool. Correlations between gradients in bile acid concentrations and microbial abundance predicted species that altered the bile acid pool through deconjugation. Furthermore, microbially conjugated bile acid concentrations exhibited amino acid-dependent trends that were not apparent in stool. Overall, non-invasive, longitudinal profiling of microorganisms, proteins and bile acids along the intestinal tract under physiological conditions can help elucidate the roles of the gut microbiome and metabolome in human physiology and disease.