*Purpose: Ischemia-reperfusion injury (IRI) is a major risk factor in orthotopic liver transplantation (OLT). As host microbiota may associate with health or disease, antibiotics (Abx) and fecal microbiota transplantation (FMT) can manipulate microbial profile to improve the disease status. We have reported on benefits of Abx pretreatment in IRI-OLT via prostaglandin (PGE2/EP4) pathway in mice and humans. Here, we evaluated the effects of Abx/FMT on taxonomical composition and outcomes in mouse OLT recipients.

*Methods: In IRI-OLT arm (n=6/gr), mice (C57BL/6) pretreated with/without 10d course of amoxicillin (AMPC; 50g/L in drinking water) were transplanted with 18h cold-stored liver allografts (Balb/c), followed by OLT/blood sampling (6h). In FMT group (n=4/gr), AMPC-pretreated mice received adjunctive FMT from naïve donors + OLT. In fecal analysis arm, cage-controlled mice were divided into distinct groups: 1) control; 2) AMPC alone; 3) AMPC+FMT; 4) OLT. To ensure the selective effect of anaerobe bacteria on OLT function/outcomes, metronidazole (MNZ; 1g/L in drinking water for 10d) was given with/without FMT (n=4/gr).

*Results: 10-day AMPC pretreatment alleviated IRI in OLT recipients, evidenced by decreased sALT (3359±1574 vs. 7383±1501U/L, p<0.05), Suzuki's histological score, frequency of TUNEL+ cells, infiltrating neutrophils/macrophages and enhanced prostaglandin EP4 receptor expression (IHC). Adjunctive FMT restored classic hepatic IRI (sALT: 7485±847.6 U/L, p<0.05 vs. AMPC) and reduced hepatic EP4 levels. In fecal 16S q-PCR analysis, there were no changes in absolute microbiota levels between groups; while 16S rRNA-amplicon sequencing showed decreased species richness, abundance, and diversity in AMPC vs. controls (Faith’s, Shannon and Chao1; p<0.005 in each index). Adjunctive FMT abrogated this commensal microbiota pattern (p<0.005 vs. AMPC, ns vs. control); and by day 1 post-OLT microbiota was comparable with controls. Levels of Akkermansia and Bacteroidetes were increased in AMPC group vs. control; while Ruminococcaceae, Turicibacter, and Clostridium were reduced. Remarkably, MNZ pretreated OLT experienced severe IRI, with increased sALT (8853±353.5 vs. 5486±1345 U/L, p<0.05), Suzuki’s score, TUNEL+ cells, and graft-infiltrating neutrophils/macrophages, and decreased EP4 expression vs. controls. However, adjunctive FMT in MNZ-treated OLT recipients restored hepatocellular function to control levels (sALT: 4301±2145U/L, ns vs. control, p<0.05 vs. MNZ group).

*Conclusions: This study documents striking benefits of microbiota modulation in a clinically-relevant mouse OLT model; and identifies a specific microbiota genus profile as a target for novel therapeutic manipulation in IRI-OLT.

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