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

Abstract

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.

Using click chemistry to study microbial ecology and evolution

“…This can occur if some species are intrinsically less efficient at incorporating the label of choice or if there is competition for uptake with the unlabeled biomolecule. For example, EdU is unable to label all bacterial species [36], for reasons that remain unclear. In addition, both AHA and HPG are incorporated less efficiently in the presence of methionine [44], which may be a limitation in environmental or host-associated environments containing exogenous methionine.…”

“…EdU-click was combined with FACS-seq to identify the replicating cells within the murine microbiome [36]. Although the results were promising and could, for example, quantify changes in the replicating bacteria within the fecal microbiome before and after antibiotic exposure, it also highlighted potential limitations.…”