Intestinal microbiota transplantation (IMT) has proven to be an effective investigative therapy for the treatment of chronic Clostridium difficile infections (CDI). Although this approach has shown astounding success, the analysis of response during transplantation has been complicated by the inability to associate mobile elements (plasmids and phage) with their microbial hosts without culturing. These mobile elements can transfer AMR and virulence genes from donor to host, and phages may also act to remodel the gut microbiota of people with CDI.

Proximity ligation sequencing (Hi-C) is an approach which uses crosslinks generated in vivo between the host microbial genome and the genetic material of both plasmids and phage. This crosslinked chromatin undergoes paired-end sequencing and bioinformatic processing which, in addition to recovering highly complete microbial genomes, allows for the recovery of the phage-host and plasmid-host interactome.

We applied proximity ligation sequencing (ProxiMeta™️) to a time course of samples from a large cohort of patients enrolled in a clinical trial using IMT for CDI. We were able to recover a complex network of interactions between microbes and mobile elements, and uncovered the dynamics of how these networks are remodeled during the process of transplantation and recovery. We demonstrated that this method can be used to screen donors for potentially dangerous AMR and virulence genes. We also identified several phage which may play a key role in the process of microbiome remodeling. Finally, we identify phage which target AMR-containing microbes, suggesting a path towards the discovery of novel therapeutic phage.