Fecal microbiota transplantation (FMT) is a therapeutic intervention for inflammatory diseases of the gastrointestinal tract, but its clinical mode of action and subsequent microbiome dynamics remain poorly understood. Here we analyzed metagenomes from 316 FMTs, sampled pre and post intervention, for the treatment of ten different disease indications. We quantified strain-level dynamics of 1,089 microbial species, complemented by 47,548 newly constructed metagenome-assembled genomes. Donor strain colonization and recipient strain resilience were mostly independent of clinical outcomes, but accurately predictable using LASSO-regularized regression models that accounted for host, microbiome and procedural variables. Recipient factors and donor–recipient complementarity, encompassing entire microbial communities to individual strains, were the main determinants of strain population dynamics, providing insights into the underlying processes that shape the post-FMT gut microbiome. Applying an ecology-based framework to our findings indicated parameters that may inform the development of more effective, targeted microbiome therapies in the future, and suggested how patient stratification can be used to enhance donor microbiota colonization or the displacement of recipient microbes in clinical practice.

Summary Taxonomic analysis of microbial communities is well supported at the level of species and strains. However, species can contain significant phenotypic diversity and strains are rarely widely shared across global populations. Stratifying the diversity between species and strains can identify ‘subspecies’, which are a useful intermediary. High-throughput identification and profiling of subspecies is not yet supported in the microbiome field. Here, we use an operational definition of subspecies based on single nucleotide variant (SNV) patterns within species to identify and profile subspecies in metagenomes, along with their distinctive SNVs and genes. We incorporate this method into metaSNV v2, which extends existing SNV-calling software to support further SNV interpretation for population genetics. These new features support microbiome analyses to link SNV profiles with host phenotype or environment and niche-specificity. We demonstrate subspecies identification in marine and fecal metagenomes. In the latter, we analyze 70 species in 7524 adult and infant subjects, supporting a common subspecies population structure in the human gut microbiome and illustrating some limits in subspecies calling. Availability and implementation Source code, documentation, tutorials and test data are available at https://github.com/metasnv-tool/metaSNV and https://metasnv.embl.de. Supplementary information Supplementary data are available at Bioinformatics online.

Liver-generated plasma Apolipoprotein E (ApoE)-containing lipoproteins (LPs) (ApoE-LPs) play central roles in lipid transport and metabolism. Perturbations of ApoE can result in several metabolic disorders and ApoE genotypes have been associated with multiple diseases. ApoE is synthesized at the endoplasmic reticulum and transported to the Golgi apparatus for LP assembly; however, the ApoE-LPs transport pathway from there to the plasma membrane is largely unknown. Here, we established an integrative imaging approach based on a fully functional fluorescently tagged ApoE. We found that newly synthesized ApoE-LPs accumulate in CD63-positive endosomes of hepatocytes. In addition, we observed the co-egress of ApoE-LPs and CD63-positive intraluminal vesicles (ILVs), which are precursors of extracellular vesicles (EVs), along the late endosomal trafficking route in a microtubule-dependent manner. A fraction of ApoE-LPs associated with CD63-positive EVs appears to be co-transmitted from cell to cell. Given the important role of ApoE in viral infections, we employed as well-studied model the hepatitis C virus (HCV) and found that the viral replicase component nonstructural protein 5A (NS5A) is enriched in ApoE-containing ILVs. Interaction between NS5A and ApoE is required for the efficient release of ILVs containing HCV RNA. These vesicles are transported along the endosomal ApoE egress pathway. Taken together, our data argue for endosomal egress and transmission of hepatic ApoE-LPs, a pathway that is hijacked by HCV. Given the more general role of EV-mediated cell-to-cell communication, these insights provide new starting points for research into the pathophysiology of ApoE-related metabolic and infection-related disorders.

Clostridioides difficile is an urgent threat in hospital-acquired infections world-wide, yet the microbial composition associated with C. difficile, in particular in C. difficile infection (CDI) cases, remains poorly characterised. Here, we analysed 534 metagenomes from 10 publicly available CDI study populations. While we detected C. difficile in only 30% of CDI samples, multiple other toxigenic species capable of inducing CDI-like symptomatology were prevalent, raising concerns about CDI overdiagnosis. We further tracked C. difficile in 42,814 metagenomic samples from 253 public studies. We found that C. difficile prevalence, abundance and association with other bacterial species is age-dependent. In healthy adults, C. difficile is a rare taxon associated with an overall species richness reduction, while in healthy infants C. difficile is a common member of the gut microbiome and its presence is associated with a significant increase in species richness. More specifically, we identified a group of species co-occurring with C. difficile exclusively in healthy infants, enriched in obligate anaerobes and in species typically found in the gut microbiome of healthy adults. Overall, gut microbiome composition in presence of C. difficile in healthy infants is associated with multiple parameters linked to a healthy gut microbiome maturation towards an adult-like state. Our results suggest that C. difficile is a commensal in infants, and that its asymptomatic carriage is dependent on the surrounding microbial context.

Liver-generated plasma Apolipoprotein E (ApoE)-containing lipoproteins (LPs) (ApoE-LPs) play central roles in lipid transport and metabolism. Perturbations of ApoE can result in several metabolic disorders and ApoE genotypes have been associated with multiple diseases. ApoE is synthesized at the endoplasmic reticulum and transported to the Golgi apparatus for LP assembly; however, ApoE-LPs transport from there to the plasma membrane is largely unknown. Here, we established an integrative imaging approach based on a fully functional fluorescently tagged ApoE. We found that ApoE-LPs accumulate in CD63-positive endosomes of hepatocytes. In addition, we observed the co-egress of ApoE-LPs and extracellular vesicles (EVs) along the late endosomal trafficking route. Moreover, complexes of ApoE-LPs and CD63-positive EVs were found to be transmitted from cell to cell. Given the important role of ApoE in viral infections, we studied the hepatitis C virus (HCV) and found that the viral replicase protein NS5A is enriched in ApoE-containing intraluminal vesicles. Interaction between NS5A and ApoE is required for the efficient release of EVs containing viral RNA. These vesicles are transported along the endosomal ApoE egress pathway. Taken together, our data argue for endosomal egress and transmission of hepatic ApoE-LPs, a pathway that is hijacked by HCV. Given the more general role of EV-mediated cell-to-cell communication, these insights provide new starting points for research into the pathophysiology of ApoE-related metabolic and infection-related disorders.