Identification of Bacterial and Viral Consortia for the Production of Intestinal Microbiota-Derived Metabolites Associated with Protection in Allogeneic Stem Cell Transplantation Patients

Background: The intestinal bacteriome directly affects clinical outcome in patients undergoing allogeneic hematopoietic stem cell transplantation (allo-SCT). Besides bacteria, fungal and viral communities as well as microbiota-derived secondary metabolites play a role. Yet, it is still unclear how dynamic shifts in these three communities:

(1) Contribute to clinical outcome of allo-SCT patients,

(2) Determine intestinal levels of microbiota-derived metabolites,

(3) Are affected by microbiome alterations such as antibiotics or modulation via fecal microbiota transplantation (FMT).

Methods: We performed a prospective, longitudinal study that combined transkingdom analysis (bacterial 16S rRNA, fungal 18S rRNA, viral metagenomic sequencing) of intestinal microbial communities with targeted metabolomics by mass spectrometry in allo-SCT patients (n=78, Table 1) at two different transplantation centers (Munich and Regensburg). Our goal was to identify microbiome signatures associated with the production of intestinal microbiota-derived metabolites. Multi-omics factor analysis (MOFA)-identified microbiome signatures characterized by high metabolite expression were subsequently analyzed by metagenomic sequencing for (1) high-resolution profiling of the taxonomic community composition and (2) characterization of alterations in microbial genes and pathways during allo-SCT. Candidate microbial genes that were highly differentially abundant before and after allo-SCT were validated across all patient samples by quantitative PCR (qPCR). Levels of intestinal metabolites were correlated with clinical outcome (overall survival (OS), transplant-related mortality (TRM), incidence graft-versus-host disease (GvHD)). A graphical abstract is provided in Figure 1.

Results: By integrating bacterial, fungal and viral sequencing data with metabolite expression via MOFA, we identified two Factors comprised of bacteria and bacteriophages which explained the most variance across all patient samples and omics modalities. These Factors described a microbiome signature of metabolite-producing bacteria from the Lachnospiraceae and Oscillospiraceae families and their corresponding bacteriophages, which correlated with the intestinal production of immunomodulatory metabolites including short-chain fatty acids (SCFAs), metabolites associated with induction of type-I IFN signaling (IIMs) and secondary bile acids. A high expression of these MOFA-identified Factors before allo-SCT correlated with reduced TRM and GvHD.

By metagenomic sequencing, we observed that microbial genes associated with bacterial metabolic pathways were enriched in the identified bacterial/bacteriophage consortia. From a set of highly enriched candidate genes, we chose to validate the expression of Butyryl-coenzyme A (CoA):acetate CoA-transferase (BCoAT), a bacterial enzyme required for biosynthesis of the SCFA butyrate. By qPCR, we observed high BCoAT abundance in early as compared to late time-points across all patients samples. In line, sustained production of intestinal metabolites after allo-SCT was associated with improved OS and reduced TRM, whereas antibiotic exposure significantly impaired metabolite expression.

As outlook, we demonstrate that single taxa domination and metabolite depletion in a patient suffering from GvHD could be rescued by transfer of metabolite-producing bacterial/bacteriophage consortia via FMT. FMT led to resolution of steroid refractory GvHD and was accompanied by an increase of bacterial and viral diversity, restoration of SCFAs and IIMs and accumulation of regulatory T cells to the intestine.

Conclusions: Our study demonstrates that sustained local production of intestinal metabolites is associated with better OS, reduced TRM and less GVHD in patients receiving allo-SCT. Microbiome alterations by antibiotics or modulation by FMT can affect the identified bacterial/bacteriophage consortia, the expression of metabolite biosynthesis pathways and the intestinal production of protective microbiota-derived metabolites thereby steering clinical outcomes. Our study provides a rationale for the development of engineered metabolite-producing consortia and defined metabolite combination drugs as novel microbiome-based therapies.

Wolff:Incyte Corporation: Honoraria; Novartis: Honoraria, Research Funding; Behring: Honoraria; Sanofi: Honoraria. Bassermann:BMS: Honoraria. Holler:Maat-Pharma (Lyon France): Membership on an entity's Board of Directors or advisory committees; Pharmabiome (Zürich, CH): Membership on an entity's Board of Directors or advisory committees.