No abstract

The gut microbiome (GM) is shaped through infancy and plays a major role in determining susceptibility to chronic diseases later in life. Bacteriophages (phage) are known to modulate bacterial populations in numerous ecosystems, including the gut. However, virome data is difficult to analyse because it mostly consists of unknown viruses, i.e. viral dark matter. Here, we manually resolved the viral dark matter in the largest human virome study published to date. Fecal viromes from a cohort of 647 infants at 1 year of age were deeply sequenced and analysed through successive rounds of clustering and curation. This uncovered more than ten thousand viral species distributed over 248 viral families falling within 17 viral order-level clusters. Most of the defined viral families and orders were novel and belonged to the Caudoviricetes viral class. Bacterial hosts were predicted for 79% of the viral species using CRISPR spacers in metagenomes from the same fecal samples. While Bacteroides-infecting Crassphages were present, novel viral families were more predominant, including phages infecting Clostridiales and Bifidobacterium. Phage lifestyles were determined for more than three thousand caudoviral species. Lifestyles were homogeneous at the family level for 149 caudiviral families. 32 families were found to be virulent, while 117 families were temperate. Virulent phage families were more abundant but temperate phage families were more diverse and widespread. Together, the viral families found in this study represent a major expansion of current bacteriophage taxonomy, and the sequences have been put online for use and validation by the community.

The development of necrotizing enterocolitis (NEC), a life-threatening inflammatory bowel disease affecting preterm infants, is connected with gut microbiota dysbiosis. Using preterm piglets as a model for preterm infants we recently showed that fecal microbiota transplantation (FMT) from healthy suckling piglet donors to newborn preterm piglets decreased the NEC risk. However, in a follow-up study using donor stool from piglets recruited from another farm, this finding could not be replicated. This allowed us to study donor-recipient microbiota dynamics in a controlled model system with a clear difference in NEC phenotype. Preterm piglets (n = 38) were randomly allocated to receive control saline (CON), or rectal FMT using either the ineffective (FMT1) or the effective donor stool (FMT2). All animals were followed for four days before necropsy and gut pathological evaluation. Donor and recipient colonic gut microbiota (GM) were analyzed by 16 S rRNA gene amplicon sequencing and shotgun metagenomics. As expected, only FMT2 recipients were protected against NEC. Both FMT groups had shifted GM composition relative to CON, but FMT2 recipients had a higher lactobacilli relative abundance compared to FMT1. Limosilactobacillus reuteri and Lactobacillus crispatus strains of FMT recipients showed high phylogenetic similarity with their respective donors, indicating engraftment. Moreover, the FMT2 group had a higher lactobacilli replication rate and harbored specific glycosaminoglycan-degrading Bacteroides. In conclusion, subtle species-level donor differences translate to major changes in engraftment dynamics and the ability to prevent NEC. This could have implications for proper donor selection in future FMT trials for NEC prevention.

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