Metabolic syndrome (MetSyn), a cluster of conditions increasing cardiometabolic disease risk, affects a substantial portion of the global population. The gut microbiota, comprising bacteria, viruses, archaea, fungi, and protists, plays a role in MetSyn pathogenesis. Bacteriophages (phages), the dominant viral component, shape microbial communities through bacterial lysis or lysogeny. Previous research indicated reduced gut phageome richness and diversity in MetSyn. Given phages' ability to modulate gut bacteria and the rise of multidrug-resistant bacteria, interest in phage therapy and fecal virome transplantation (FVT) is growing. FMTs, while effective, carry risks. Sterile FFT, lacking live bacteria, offers a safer alternative. Prior studies showed FVT decreased weight gain and normalized blood glucose in obese mice. This study aimed to determine if FFT could induce similar effects in humans with MetSyn, focusing on safety and longitudinal changes in the bacteriome and phageome.

Previous research has primarily focused on the bacterial component of the gut microbiome's impact on human health, particularly in metabolic syndrome. However, the viral component, primarily bacteriophages, is also increasingly recognized for its role in shaping microbial communities and influencing human health. Studies have shown that reduced richness and diversity of the gut phageome are associated with metabolic syndrome, along with altered composition and increased inter-individual variation. The therapeutic potential of phages has also been explored, particularly in phage therapy to target specific pathogenic bacteria. However, the efficacy of using phage cocktails to beneficially alter a complete microbiome is still under investigation. Therefore, fecal virome transplantation (FVT) is receiving more attention as a potential strategy to modulate the gut microbiome for therapeutic benefit, especially given the demonstrated efficacy of FVT in murine models of obesity. The safety profile of FFT compared to FMT is also considered a significant advantage, as it reduces the risk of transferring unknown pathogenic bacteria.

This prospective, double-blind, randomized, placebo-controlled pilot study enrolled 24 individuals with MetSyn. Participants were randomized to receive either an FFT from a lean healthy donor (n=12) or a placebo (n=12). The primary outcome measure was the change in glucose metabolism, assessed via the area under the curve (AUC) for glucose excursion during an oral glucose tolerance test (OGTT) from baseline to day 28. Secondary outcomes included safety (adverse events), changes in other glucose metabolism parameters (fasting glucose, insulin, HOMA-IR, HbA1c, glucose variability from CGM), and longitudinal changes in the intestinal bacteriome and phageome. Faecal samples were collected at multiple timepoints for whole-genome shotgun (WGS) sequencing of both the bacteriome and phageome, as well as for viral-like particle (VLP) sequencing to study phage virions. The FFT was prepared by homogenizing donor stool in saline, centrifuging to remove bacteria, and filtering through a 0.2 µm membrane. The placebo was sterile saline with a brown colorant. Participants used a laxative the day before the intervention. Statistical analyses included paired Wilcoxon signed-rank test, Mann-Whitney U test, PERMANOVA, ANCOM-BC, and Spearman correlation.

All 24 participants completed the study. The FFT was well-tolerated, with only mild gastrointestinal adverse events reported, more frequently in the FFT group than the placebo group (though not statistically significant). There were no significant differences in overall glucose metabolism (AUC) between the FFT and placebo groups at day 28, nor significant changes in fasting glucose, insulin, HOMA-IR, and HbA1c. However, glucose variability (time in target range of 3.9-10 mmol/L) showed a nominal significant improvement in the FFT group post intervention (though this disappeared after multiple testing correction). Phage virion composition differed significantly between groups on day 2 post-intervention, with 216 differentially abundant viral populations (VPs). Six bacterial host species showed significant enrichment among these VPs, and analysis of phage-host interactions revealed a negatively correlated relationship (indicating antagonism) between phage and bacterial abundance in the FFT group on days 0-2, compared to a positive correlation (protagonism) in the placebo group. This difference was less pronounced on days 2-28 and absent on days 0-28, suggesting a transient effect. Bacterial and viral diversity remained stable after FFT.

This study provides evidence that FFT is a safe method for transiently altering the gut microbiota in individuals with MetSyn. Although the primary outcome of improved glucose metabolism was not achieved, the observed shift in phage virion composition and phage-microbe interactions on day 2 post-FFT warrants further investigation. The transient nature of these effects suggests that optimized FFT protocols with a better-defined virome, potentially through pooling of donor phages and improved donor-recipient matching, may be necessary to achieve more sustained changes in gut microbiota and metabolic parameters. The lack of significant changes in overall glucose metabolism might be attributed to the small sample size, heterogeneity within the MetSyn population, and the impact of the laxative pre-treatment.

This double-blind, randomized, placebo-controlled trial demonstrates the safe administration of a sterile fecal filtrate to individuals with metabolic syndrome. While significant improvement in overall glucose metabolism wasn't observed, the study reveals a transient alteration of the gut phageome and phage-microbe interactions. Future research should focus on optimizing FFT protocols, including improved donor-recipient matching, to enhance efficacy and investigate longer-term effects. Exploring targeted phage therapies against specific bacteria involved in MetSyn pathogenesis is also warranted.

The study's limitations include the small sample size, which may have limited the power to detect differences in glucose metabolism, and the heterogeneity within the MetSyn population. The use of a laxative before the intervention might have also affected the results. The FFT preparation method, while safe, might not have delivered a sufficiently concentrated or diverse phageome for sustained effect. The focus on dsDNA phages excludes potential contributions from other phage types. Finally, the study's generalizability is limited by the inclusion of only European Dutch subjects.