The composition of the intestinal bacterial community is well described, but recent research suggests that the metabolism of these bacteria plays a larger role in health than which species are present. One fundamental aspect of gut bacterial metabolism that remains understudied is bacterial replication. Indeed, there exist few techniques which can identify actively replicating gut bacteria. In this study, we aimed to address this gap by adapting 5-ethynyl-2’-deoxyuridine (EdU) click chemistry (EdU-click), a metabolic labeling method, coupled with fluorescence-activated cell sorting and sequencing (FACS-Seq) to characterize replicating gut bacteria. We first used EdU-click with human gut bacterial isolates and show that many of them are amenable to this technique. We then optimized EdU-click and FACS-Seq for murine fecal bacteria and reveal that Prevotella UCG-001 and Ileibacterium are enriched in the replicating fraction. Finally, we labeled the actively replicating murine gut bacteria during exposure to cell wall-specific antibiotics in vitro . We show that regardless of the antibiotic used, the actively replicating bacteria largely consist of Ileibacterium , suggesting the resistance of this taxon to perturbations. Overall, we demonstrate how combining EdU-click and FACSeq can identify the actively replicating gut bacteria and their link with the composition of the whole community in both homeostatic and perturbed conditions. This technique will be instrumental in elucidating in situ bacterial replication dynamics in a variety of other ecological states, including colonization and species invasion, as well as for investigating the relationship between the replication and abundance of bacteria in complex communities.

The composition of the intestinal bacterial community is well described, but recent research suggests that the metabolism of these bacteria plays a larger role in health than which species are present. One fundamental aspect of gut bacterial metabolism that remains understudied is bacterial replication. Indeed, there exist few techniques which can identify actively replicating gut bacteria. In this study, we aimed to address this gap by adapting 5-ethynyl-2′-deoxyuridine (EdU) click chemistry (EdU-click), a metabolic labeling method, coupled with fluorescence-activated cell sorting and sequencing (FACS-Seq) to characterize replicating gut bacteria. We first used EdU-click with human gut bacterial isolates and show that many of them are amenable to this technique. We then optimized EdU-click and FACS-Seq for murine fecal bacteria and reveal that Prevotella UCG-001 and Ileibacterium are enriched in the replicating fraction. Finally, we labelled the actively replicating murine gut bacteria during exposure to cell wall-specific antibiotics in vitro. We show that regardless of the antibiotic used, the actively replicating bacteria largely consist of Ileibacterium, suggesting the resistance of this taxon to perturbations. Overall, we demonstrate how combining EdU-click and FACSeq can identify the actively replicating gut bacteria and their link with the composition of the whole community in both homeostatic and perturbed conditions. This technique will be instrumental in elucidating in situ bacterial replication dynamics in a variety of other ecological states, including colonization and species invasion, as well as for investigating the relationship between the replication and abundance of bacteria in complex communities.

Manipulations to the microbiota may serve as a potential treatment for obesity. Oligofructose (OFS), a nondigestible carbohydrate, beneficially alters the distal gut microbiome and results in decreased food intake and bodyweight, although the exact mechanisms remain unknown. Despite the importance of small intestinal (SI) gut-brain nutrient feedback mechanisms in regulating food intake and energy homeostasis, no studies have directly examined whether OFS can improve SI gut-brain signaling, potentially via changes in the SI microbiota. Given recent evidence highlighting the importance of SI microbiota on regulating metabolic homeostasis, we tested the hypothesis that OFS specifically improves SI lipid-sensing mechanisms controlling food intake, via manipulation of SI microbiota. Before any significant changes in body weight are observed, acute (3d) treatment with OFS in high fat (HF)-fed rats results in a significant suppression of 2hr food intake following an intralipid or liquid meal SI infusion (6kcal) compared to HF alone, likely due to a restored gut to brain signal. OFS treatment also increased jejunal CD36 gene and protein expression, which is known to mediate lipid-induced release of GLP-1. As such, OFS-treated rats exhibited significantly increased portal GLP-1 levels and c-fos, a marker neuronal activation, in the NTS of the hindbrain following intralipid SI infusion, compared to HF-fed rats. Importantly, transplant of SI microbiota from 3d OFS HF-fed donors into HF-fed rats recapitulated these results, demonstrating the importance of OFS-induced SI microbiota changes in restoring nutrient-induced gut brain signaling. These results highlight the importance of the small intestinal microbiota in energy regulation and identify a potential role of the small intestine and small intestinal nutrient sensing in the beneficial effects of OFS. Disclosure S.N. Weninger: None. E. Beauchemin: None. A.I.L. Lane: None. R. Meyer: None. F. Duca: None. Funding Arizona Biomedical Research Commission (NIA17-7401)