**1. Introduction**

Type 2 diabetes mellitus (T2DM) continues to be a worldwide clinical challenge. The gut microbiota plays an important role in determining host metabolic health and has been associated with T2DM. Studies comparing the composition and function of the fecal microbiota from groups who had T2DM, impaired glucose tolerance, or normal glucose tolerance have reported distinct bacterial compositions. For example, *Roseburia* and *Faecalibacterium prausnitzii* are differentially enriched in T2DM [1]. Additionally, decreases in Bacteroidetes and increases in Actinobacteria and Firmicutes are associated with obesity [2,3]. Studies in germ-free mice show that transplantation of the gut microbiota from metabolically

**Citation:** Bustamante, J.-M.; Dawson, T.; Loeffler, C.; Marfori, Z.; Marchesi, J.R.; Mullish, B.H.; Thompson, C.C.; Crandall, K.A.; Rahnavard, A.; Allegretti, J.R.; et al. Impact of Fecal Microbiota Transplantation on Gut Bacterial Bile Acid Metabolism in Humans. *Nutrients* **2022**, *14*, 5200. https://doi.org/10.3390/ nu14245200

Academic Editor: Abeer M. Mahmoud

Received: 31 October 2022 Accepted: 2 December 2022 Published: 7 December 2022

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

healthy vs. metabolically impaired donors transfers these metabolic phenotypes, pointing to a causative role for the gut microbiota in the pathogenesis of metabolic disease [4,5]. Therefore, the gut microbiome is an attractive target for the treatment and prevention of T2DM.

Fecal microbiota transplantation (FMT) is a potential method to target the gut microbiome for T2DM treatment and prevention [6]. FMT has been shown to successfully treat microbiota-related dysfunction, with the treatment of *Clostridioides difficile* infection being the most notable example of its successful therapeutic use [7,8]. To test the potential utility of FMT for the treatment of metabolic disease, our group previously studied patients with obesity, without metabolic impairment, treated with FMT or placebo. FMT did not induce weight loss but did successfully colonize the gastrointestinal tract of recipients and slowed the development of glucose intolerance compared with placebo, as assessed by mixed meal tolerance testing [9]. The mechanisms for this microbially induced improvement in glucose tolerance are unknown. However, a key mechanism by which the gut microbiota influences host metabolic health is through the production of metabolites, such as short-chain fatty acids (SCFAs) and unconjugated and secondary bile acids. Although SCFA concentrations were not altered by FMT, FMT increased gut bacterial bile acid metabolism compared to placebo resulting in a change in the bile acid profile that mirrored that of the lean donor [10].

Bile acids are a class of bioactive metabolites that signal through bile acid receptors, such as FXR and TGR5, to improve metabolic health. Bile acids are primarily metabolized by the liver and the gut microbiota. Primary bile acids are produced in the liver from cholesterol and are conjugated with taurine or glycine prior to secretion into the gut lumen. Primary bile acids are converted into secondary bile acids, deoxycholic acid (DCA) and lithocholic acid (LCA), by gut bacteria. Bile acids vary in their affinity for bile acid receptors. Therefore, alterations in the bile acid profile can influence metabolic health by altering bile acid receptor signaling. In particular, DCA and LCA are the strongest ligands for TGR5 [11–14]. Indeed, studies investigating the impact of gut microbiota on metabolic disease often identify gut bacterial bile acid production as a key mechanistic mediator. For example, dietary fiber supplementation has been reported to enhance gut bacterial 6-α-hydroxylation to improve metabolic phenotypes in mice [15]. Furthermore, recent work reports that enhancing gut bacterial bile acid deconjugation through the use of genetically modified microbes improves metabolic parameters in mice [16].

A key pathway in gut bacterial bile acid metabolism is the conversion of conjugated primary bile acids to secondary bile acids through deconjugation followed by 7-αdehydroxylation. Primary bile acids are first deconjugated by the enzyme bile salt hydrolase (BSH) [17,18]. BSH expression has been identified across all major bacterial divisions and archaeal species in the gut, and elevations in BSH activity improve metabolic outcomes [19]. Furthermore, BSH activity enhances bacterial survival [19]. Therefore, BSH may also be a key determinant of the efficacy by which probiotics and FMT are able to successfully colonize the host [20]. However, the regulation of BSH expression is poorly understood. Unconjugated bile acids are converted to secondary bile acids through 7-α-dehydroxylation, which is a multi-step process that is less widely dispersed throughout the gut microbiome relative to BSH [17,18]. Nevertheless, the gut bacterial species and genes responsible for 7-α-dehydroxylation are still incompletely defined.

Research suggests FMT increases gut microbial diversity and the abundance of beneficial bacteria. Indeed, we found that patients who received FMT show sustained shifts in gut microbiota profiles toward those of the donor, as determined by 16S rRNA gene sequencing. Additionally, bile acid profiles resembled that of the donor [10]. Importantly, this shift in bile acid profile also coincided with a slowing of glycemic impairment compared with placebo [9]. The results from this clinical pilot, which evaluated the effectiveness of FMT in obese metabolically healthy patients, provide an ideal study set to identify gut bacteria involved in gut bacterial bile acid metabolism. Therefore, we assessed the impact of FMT on gut bacterial composition by metagenomics to better understand the dynamic alterations induced by FMT. Furthermore, we assessed the correlation between bacterial abundance

determined by metagenomics with bile acid levels, assessed in the same samples, to identify putative bacterial species that may contribute to gut microbial bile acid metabolism.
