Overcoming donor variability and risks associated with fecal microbiota transplants through bacteriophage-mediated treatments

Abstract

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.

Transfer of modified fecal viromes improve blood glucose regulation and alleviates symptoms of metabolic dysfunction-associated fatty liver disease in an obesity male mouse model

“…Solvent/detergent treatment (approved by the World Health Organization for treating blood plasma 35 ) was used to inactivate enveloped viruses 36 (FVT-SDT), a compound (pyronin Y) that specifically binds to RNA 37,38 was applied to inactivate RNA viruses (FVT-PyT), and an optimized chemostat fermentation of intestinal inoculum aimed at removing the eukaryotic viruses by dilution 31 (FVT-ChP). We have also demonstrated that the modified FVT-SDT and FVT-ChP showed promising results in treating C. difficile infections in a mouse model 30 , which represents a simple disease etiology mainly caused by the toxin-producing C. difficile 39,40 . The more complex GM associated diet-induced obesity model 41 was included in this study to investigate whether the same modified FVTs 30 could be used to improve phenotypes from these two very different disease etiologies through phage-mediated restoration of a dysbiotic GM.…”

“…We have also demonstrated that the modified FVT-SDT and FVT-ChP showed promising results in treating C. difficile infections in a mouse model 30 , which represents a simple disease etiology mainly caused by the toxin-producing C. difficile 39,40 . The more complex GM associated diet-induced obesity model 41 was included in this study to investigate whether the same modified FVTs 30 could be used to improve phenotypes from these two very different disease etiologies through phage-mediated restoration of a dysbiotic GM. Only male mice were included, since female mice are highly protected against diet-induced obesity 42 .…”

“…However, the centrifugation and filtration steps used in preparing FVT cannot separate phages from eukaryotic viruses due to their similar sizes, that together with donor variability, makes widespread use of FVT to treat obesity and type-2 diabetes unlikely. In two recent studies, we aimed to enhance the safety of FVT by developing methodologies that selectively inactivate 30 (not yet peer-reviewed) or remove the eukaryotic viruses from the fecal matrix 31 (not yet peer-reviewed) while preserving an active enteric phage community. To achieve this, we utilized the differences in key characteristics between eukaryotic viruses and phages 30,31 ; most eukaryotic viruses are enveloped RNA viruses 32,33 that only infect eukaryotic cells, and most phages are non-enveloped DNA viruses 33,34 infecting only bacteria.…”

“…In two recent studies, we aimed to enhance the safety of FVT by developing methodologies that selectively inactivate 30 (not yet peer-reviewed) or remove the eukaryotic viruses from the fecal matrix 31 (not yet peer-reviewed) while preserving an active enteric phage community. To achieve this, we utilized the differences in key characteristics between eukaryotic viruses and phages 30,31 ; most eukaryotic viruses are enveloped RNA viruses 32,33 that only infect eukaryotic cells, and most phages are non-enveloped DNA viruses 33,34 infecting only bacteria. Solvent/detergent treatment (approved by the World Health Organization for treating blood plasma 35 ) was used to inactivate enveloped viruses 36 (FVT-SDT), a compound (pyronin Y) that specifically binds to RNA 37,38 was applied to inactivate RNA viruses (FVT-PyT), and an optimized chemostat fermentation of intestinal inoculum aimed at removing the eukaryotic viruses by dilution 31 (FVT-ChP).…”

“…Our main objective was therefore to screen whether the differently modified FVTs 30,31 had the potential to alleviate symptoms on co-morbidities of metabolic syndrome as a safer and/or more reproducible alternative to the unmodified FVT that we previously have used in a similar diet-induced obesity model 25 .…”