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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., (© 2022. The Author(s).)