Highlights d Stunted children have distinct gut phage communities relative to non-stunted ones d In vitro, phages regulate bacteria isolated from children younger than 23 months d Non-stunted children harbor more temperate phages, irrespective of age d Bacteria in stunted children have more traits linked to human disease and metabolism

From digestion to pathogen resistance and immune system development, the gut microbiota and its collection of microbial genes are redefining what it means to be human. Despite tremendous advances in this field, there is still a limited understanding of how microbial metabolism in the gut impacts human health, which precludes the development of microbiota-targeted therapies. In this article, we discuss the increasing evidence emphasizing the importance of bacterial metabolism in the gut and discuss its intricate links with diet and pharmaceutical compounds leading to altered therapeutic outcomes. We also detail how applying and testing microbial ecology hypotheses will be crucial to fully understand the therapeutic potential of this host-associated community. Going forward, functional and mechanistic studies combining biomedical research, ecology, bioinformatics, statistical modeling, and engineering will be key in our pursuit of personalized medicine.

Changes in bacterial diversity in the human gut have been associated with many conditions, despite not always reflecting changes in bacterial activity. Methods linking bacterial identity to function are needed for improved understanding of how bacterial communities adapt and respond to their environment, including the gut. Here, we optimized bioorthogonal non-canonical amino acid tagging (BONCAT) for the gut microbiota and combined it with fluorescently activated cell sorting and sequencing (FACS-Seq) to identify the translationally active members of the community. We then used this novel technique to compare with other bulk community measurements of activity and viability: relative nucleic acid content and membrane damage. The translationally active bacteria represent about half of the gut microbiota, and are not distinct from the whole community. The high nucleic acid content bacteria also represent half of the gut microbiota, but are distinct from the whole community and correlate with the damaged subset. Perturbing the community with xenobiotics previously shown to alter bacterial activity but not diversity resulted in stronger changes in the distinct physiological fractions than in the whole community. BONCAT is a suitable method to probe the translationally active members of the gut microbiota, and combined with FACS-Seq, allows for their identification. The high nucleic acid content bacteria are not necessarily the protein-producing bacteria in the community; thus, further work is needed to understand the relationship between nucleic acid content and bacterial metabolism in the human gut. Considering physiologically distinct subsets of the gut microbiota may be more informative than wholecommunity profiling.

Most studies on the gut microbiome focus on the composition of this community and how it changes during disease. However, how the community transitions from a healthy state to one associated with disease is currently unknown.

While the diversity of the human gut microbiota is becoming increasingly well characterized, bacterial physiology is still a critical missing link in understanding how the gut microbiota may be implicated in disease. The current best practice for studying bacterial physiology involves the immediate storage of fecal samples in an anaerobic chamber. This reliance on immediate access to anaerobic chambers greatly limits the scope of sample populations that can be studied. Here, we assess the effects of short-term oxygen exposure on gut bacterial physiology and diversity. We use relative nucleic acid content and membrane integrity as markers of bacterial physiology, and 16S rRNA gene amplicon sequencing to measure bacterial diversity. Samples were stored for up to 6 h in either ambient conditions or in anoxic environments created with gas packs or in an anaerobic chamber. Our data indicate that AnaeroGen sachets preserve bacterial membrane integrity and nucleic acid content over the course of 6 h similar to storage in an anaerobic chamber. Short-term oxygen exposure increases bacterial membrane permeability, without exceeding inter-individual differences. As oxygen exposure remains an important experimental consideration for bacterial metabolism, our data suggest that AnaeroGen sachets are a valid alternative limiting loss of membrane integrity for short-term storage of samples from harder-to-access populations.