Fecal microbiota transplantation (FMT) from a healthy donor to recurrent C. difficile infection (CDI) patients has proven efficient in curing the disease, possibly through bacteriophage-mediated (phages) modulation of the gut microbiome landscape. Fecal virome transplantation (FVT, sterile filtrated donor feces) has also been shown efficient for treating the disease. FVT has the advantage over FMT that no bacteria are transferred, but FVT does not exclude the risk of transferring eukaryotic viruses. We aimed to develop methodologies to obtain safer FVT by removing and/or inactivating eukaryotic viruses, while maintaining an active phage community. Donor feces were used as inoculum for a chemostat-fermentation to remove eukaryotic viruses by dilution (FVT-ChP). FVT solutions underwent solvent-detergent treatment to inactivate enveloped viruses (FVT-SDT) and pyronin-Y treatment to block the replication of RNA-viruses (FVT-PyT). The efficacy of these treatments was assessed in a CDI mouse model and compared with untreated FVT (FVT-UnT), FMT, and saline-treatment as controls. Intriguingly, 8/8 mice receiving FVT-SDT did not reach the humane endpoints until planned euthanization and expressed limited symptoms of CDI. While 5/7 saline treated mice reached the humane endpoint. Compared to the saline treatment, lower C. difficile abundance (p<0.005) in the FVT-SDT-treated mice suggested that the intervention had hampered C. difficile colonization. The mice receiving FVT-ChP and FVT-UnT tended to express alleviated CDI symptoms compared to the saline control. This proof-of-concept study may constitute the initial step of developing therapeutic tools that targets a broad spectrum of gut-related diseases and thereby substituting FMT with a safer phage-mediated therapies.

Introduction: Recent evidence suggest a link between gut microbiome dysbiosis and metabolic syndrome, including type-2-diabetes, obesity, and non-alcoholic fatty liver disease (NAFLD). Fecal microbiota transplantation (FMT) has been explored as a way to restore a healthy gut microbiome in obese patients but poses safety concerns. The wide use of FMT is limited by safety concerns about transferring the entire fecal microbiome from one individual to another. Fecal virome transplantation (FVT) is a safer alternative that transfers bacteriophages without bacterial transfer, but still carries the risk of eukaryotic virus infection. Therefore, a safer and more effective tool for modulating gut microbiome is needed. Methods: We explored the potential of implementing three alternative FVT techniques with increased safety established in a recent study (eukaryotic viruses were either eliminated or inactivated) to ameliorate symptoms associated with a diet-induced obesity mouse model. Male mice were fed with an ad libitum high-fat diet before being euthanized (23 weeks of age) and received the different FVT treatments twice with one week of interval. Body weight was measured, oral glucose tests were performed, feces were sampled frequently, and liver, fat pads, mesenteric lymph node, and blood serum were sampled at termination of study. Results: FVT treatments had no effect on weight gain or the amount of epididymal white adipose tissue. Mice given regular untreated FVT (FVT-UnT) had a significant (p < 0.05) drop in the pathological score of their liver tissue when compared to HFD-control mice. Mice treated with a chemostat propagated fecal virome (FVT-ChP, eukaryotic viruses eliminated by dilution) improved their blood glucose regulation significantly (p < 0.05) compared to HFD-control mice. Gut microbiome analysis of both the bacterial and viral component suggested that bacteriophage-mediated modulation of the gut microbiome could be a driving factor for the observed effects. Conclusions: These results may lay the ground to develop safer bacteriophage-based therapeutic tools to restore the dysbiotic gut microbiome associated with metabolic syndrome.