Actively replicating gut bacteria identified by 5-ethynyl-2'-deoxyuridine (EdU) click chemistry and cell sorting.

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 UCG-001 and are enriched in the replicating fraction. Finally, we labeled the actively replicating murine gut bacteria during exposure to cell wall-specific antibiotics . We show that regardless of the antibiotic used, the actively replicating bacteria largely consist of , 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 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.