Fecal microbiota transplant (FMT) is a promising, novel intervention to promote restitution and repair of colonic dysbiosis. However, the appropriateness of FMT to fully reconstitute all parts of the gut must be questioned given that most preparations are comprised of colonic anaerobic microbes not indigenous or fit to inhabit the small intestine. Mismatches between regional host gut ecosystems and their microbiota could have adverse consequences to the host. We hypothesized that colonic microbiota will not properly restore the microbiome of the small intestine (and vice versa) and that this will have both short- and long-term regional and systemic consequences on immunity and metabolism. We treated male and female SPF (specific pathogen free) mice with antibiotics and performed jejunal, cecal, or fecal microbiota transplant (JMT, CMT, FMT, respectively) and compared them to their saline or no antibiotic controls (n>7 per group, 1 and 3 months post transplant) (Fig.1). JMT and FMT resulted in distinct, altered regional gut microbiota membership and function (P<0.001, PERMANOVA and Mann-Whitney). Importantly, these microbial changes corresponded to distinct shifts in circulating and regional gut metabolite pools, as well as intestinal and hepatic transcriptional programs that persisted up to three months. Whereas JMT primarily impacted host metabolic pathways, FMT largely affected immune pathways – highlighting the extraordinary and dichotomous impact of these two donor microbiota populations (Fig.2). We found that small and large bowel microbes influence intestinal mucosal identity, whereby regional microbes drove marker gene expression and function of the jejununal and colonic mucosa. Lastly, energy balance, respiratory exchange ratios, locomotor activity, glucose tolerance, and insulin resistance were shifted in a transplant-dependent manner revealing the global, metabolic impacts of regional gut microbial mismatches. Together, these data draw attention to the impact of regional mismatches between microbiota and ecosystem on global host physiology, immunity, and metabolism. This study addresses potential limitations and unintended consequences of current FMT practices and leads to a rethinking of strategies for the development of future live biotherapeutic products (LBPs) for clinical practice.

<b>Fig1.</b> <b>Graphical Abstract. </b>Small and large bowel microbial transplants (JMT and FMT, respectively) were performed in post antibiotic C57Bl6 male and female mice and compared to their no antibiotic and saline controls after 1 and 3-months (n<u>></u>7 per group). Mice demonstrated significant differences in energy balance and intestinal and hepatic metabolism, microbial engraftment, and metabolite pools.

Fig1. Graphical Abstract. Small and large bowel microbial transplants (JMT and FMT, respectively) were performed in post antibiotic C57Bl6 male and female mice and compared to their no antibiotic and saline controls after 1 and 3-months (n>7 per group). Mice demonstrated significant differences in energy balance and intestinal and hepatic metabolism, microbial engraftment, and metabolite pools.

<b>Fig2. Hepatic metabolic pathways are enriched in JMT-treated mice whereas immune pathways are enriched in FMT-treated mice.</b> Male C57Bl6 mice were treated for 14 days with antibiotics, followed by a 2-day recovery period, and then given JMT, CMT, FMT or sterile saline via oral gavage. One-month post-MT, hepatic samples were subjected to RNAseq. Differences in gene transcripts are depicted in the PCA plot in <b>A</b> and KEGG pathway analysis shown in <b>B</b> (n=7 per group).

Fig2. Hepatic metabolic pathways are enriched in JMT-treated mice whereas immune pathways are enriched in FMT-treated mice. Male C57Bl6 mice were treated for 14 days with antibiotics, followed by a 2-day recovery period, and then given JMT, CMT, FMT or sterile saline via oral gavage. One-month post-MT, hepatic samples were subjected to RNAseq. Differences in gene transcripts are depicted in the PCA plot in A and KEGG pathway analysis shown in B (n=7 per group).

Alan Tan

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