The gut microbiota plays a central role in host metabolism, influencing digestion, immune function, and susceptibility to disease (Krishnan et al., 2015). Among the many microbial processes within the gut, bile acid (BA) metabolism is particularly significant due to its involvement in lipid absorption, intestinal homeostasis, and the regulation of metabolic disorders (Guan et al., 2022; Wang et al., 2024b). BAs, synthesized in the liver, undergo extensive microbial modifications in the gut, which not only modulate their chemical properties but also reshape host metabolic pathways (Ramírez-Pérez et al., 2017).

While BA metabolism has been extensively studied in model organisms (Staley et al., 2017; Mohanty et al., 2024), much less is known about these microbial functions in wild animals. In particular, the gut microbiomes of wild rodents, an ecologically diverse and abundant group, remain underexplored. These animals offer a unique opportunity to study microbiota-driven metabolic processes in natural settings (Rosshart et al., 2019). Unlike their laboratory counterparts, wild rodents are exposed to variable diets, environments, and microbial landscapes (Rosshart et al., 2017; Schmidt et al., 2019), all of which can shape distinct microbiome compositions and functional capacities, including BA metabolism. Bridging this gap is essential for advancing our understanding of microbiome diversity and its influence on host physiology beyond controlled experimental systems.

Moreover, the gut microbiota is highly responsive to pathogenic infections, which can disrupt microbial balance and alter host metabolism (Vich Vila et al., 2018; Trzebny et al., 2023). In this context, it is important to note that laboratory mice maintained under controlled, pathogen-free conditions exhibit markedly different immune responses from their wild counterparts (Rosshart et al., 2017; Beura et al., 2016; Mair et al., 2021), largely because the latter inhabit complex natural environments where coinfections are common and many sources of variation present in nature are eliminated in the laboratory. Among the pathogens relevant to such natural settings, one such notable example is Enterocytozoon bieneusi, a microsporidian parasite, has been shown to significantly reshape gut microbial communities with potential consequences for host health (López-Carvallo et al., 2022). However, the effects of E. bieneusi-induced microbiota shift on BA metabolism remain largely uncharacterized, especially in wild rodent populations. Investigating these dynamics may uncover novel interactions between pathogens, microbiota, and host metabolic pathways.

In this study, we performed a genome-resolved metagenomic analysis of the gut microbiota in wild rodents, with a specific focus on microbial taxa and functional genes involved in bile acid metabolism. Leveraging a high-quality genomic dataset, we characterized microbial diversity, taxonomic structure, and key BA-related pathways, while also assessing the impact of E. bieneusi infection on these microbial communities. Our findings provide new insights into the ecological and metabolic roles of the wild rodent gut microbiome and highlight the complex interplay between host, microbiota, and pathogen in shaping metabolic function and health outcomes.

This study provides new insights into the gut microbiota of wild rodents, highlighting both their extensive microbial diversity and the specialized metabolic pathways involved in BA transformation. Analysis of 5,208 high-quality microbial genomes revealed distinctive compositional patterns, particularly the dominance of Muribaculaceae, with the genus CAG-485 emerging as a central contributor to BA metabolism. These findings indicate that wild rodents harbor highly adapted microbial consortia capable of modulating host metabolic processes and underscore the importance of further research into the physiological consequences of microbial BA transformations.

Metagenomic sequencing enabled comprehensive functional profiling of the gut microbiome, overcoming the taxonomic and functional limitations of 16S rRNA sequencing (Claesson et al., 2010; Kennedy et al., 2014). This approach facilitated direct inference of microbial functions and enabled metagenome-wide association studies linking microbiome structure to host phenotypes and disease (Wang et al., 2018; Chaston et al., 2014). In this study, the dominant phyla were Bacillota_A, Bacteroidota, and Actinomycetota, with Lachnospiraceae, Muribaculaceae, and Ruminococcaceae as the most abundant families. These taxonomic patterns likely reflect adaptations to ecological niches and diet (Rinninella et al., 2019; Couch et al., 2021; Ogasawara et al., 2023).

Primary BAs are synthesized in the liver and subsequently modified gut microbiota into secondary BAs (Ridlon et al., 2006). Using metagenomic data, we identified 10,051 genes across 5,208 genomes involved in BA transformation, including deconjugation, oxidation, and dihydroxylation processes (Guzior and Quinn, 2021; Ridlon et al., 2016). The microbial community involved in BA metabolism was dominated by Bacillota_A (50.78%), followed by Bacteroidota (25.03%) and Actinomycetota, with key roles played by Lachnospiraceae, Muribaculaceae, and Oscillospiraceae. These taxa are essential to BA metabolism and may influence host immunity and inflammatory responses (Cai et al., 2022; Lloyd-Price et al., 2019).

Gut microbiota-mediated BA metabolism affects host lipid digestion, cholesterol regulation, and multiple signaling pathways (Yang et al., 2021). A pivotal reaction in this pathway is the hydrolysis of conjugated BAs, catalyzed by bile salt hydrolases (Ridlon and Gaskins, 2024). While BSH activity has traditionally been associated with Firmicutes (Kisiela et al., 2012), our data reveal a broader taxonomic distribution, with significant contributions from Bacteroidota and Bacillota_A. BSH genes were identified in genera including Lactobacillus, Bifidobacterium, Clostridium, Bacteroides and Enterococcus (Song et al., 2019). Interestingly, 2,818 BSH-encoding genomes were found in wild rodents, many affiliated with CAG-485 and CAG-873 (Muribaculaceae and Lachnospiraceae, respectively), emphasizing their central role in modulating the host bile acid pool.

Microbes encoding 7α-HSDH, involved in the oxidation of deconjugated BAs, were less abundant but still critical (Funabashi et al., 2020). We identified 609 such genomes, primarily from Bacteroidota (Ferrandi et al., 2012). In contrast, only 34 genomes encoded baiB, a key gene in secondary BA synthesis (Mihalik et al., 2002), suggesting that late-stage BA transformation is limited to a small subset of taxa. These observations highlight the broad yet uneven distribution of BA metabolic capabilities and reinforce the ecological importance of these microbial pathways (Lin et al., 2023).

BA metabolism varies widely across host species due to differences in host physiology and environmental exposure (Thakare et al., 2018; Zhang et al., 2022). Comparative analyses revealed distinct patterns in BA metabolic gene profiles between wild rodents and other species (humans, pigs, chickens, and laboratory mice), reflecting co-evolution between microbial communities and host-specific bile acid compositions (Sinha et al., 2020; Sayin et al., 2013). For example, bacterial 7β-HSDHs mediate the conversion of 7-oxo-lithocholic acid to ursodeoxycholic acid, a bile acid with therapeutic applications (Lee et al., 2013; Ferrandi et al., 2012; Liu et al., 2011). Interestingly, the 7β-HSDH gene was absent in laboratory mice, highlighting divergences in BA pathways likely driven by domestication and constrained microbial diversity (Wei et al., 2020; Ma et al., 2020). While laboratory mice are foundational to gut microbiota research, their controlled environments may constrain microbial diversity and functionality (Hanski et al., 2024; Rosshart et al., 2017), limiting their suitability as models for BA-related studies. These findings advocate for the increased use of wild-type mice as more ecologically relevant models in microbiome and bile acid research.

Although dietary differences undoubtedly shape gut microbial communities, recent studies have demonstrated that host genotype and ecological context exert profound influences on microbial function independent of diet (Rothschild et al., 2018; Maurice et al., 2015). Moreover, research on wild versus captive animals has revealed consistent shifts in microbial diversity and metabolic potential due to domestication and environmental constraints (Clayton et al., 2016; Reese and Dunn, 2018). Wild rodents, living under natural ecological conditions and exposed to diverse microbial and dietary inputs, exhibit distinct BA-transforming microbial profiles compared to laboratory mice. These differences likely reflect long-term host-microbe co-adaptation and ecological pressures rather than dietary influences alone.

Bile tolerance is a vital trait that enables microbial communities to survive and function effectively within the intestinal environment (Lin et al., 2023). In this study, functional profiling of BSH-positive genomes, particularly from the dominant CAG-485 lineage, revealed key metabolic traits that may confer bile resistance and competitive advantages. The enrichment of genomes in glycoside hydrolase (GH) families GH13 and GH16, essential for carbohydrate metabolism, suggests that BSH-positive microbes possess an expanded capacity for energy acquisition and niche adaptation. Notably, GH63 enzymes, capable of hydrolyzing α-glucosidic linkages in host-derived glycans and dietary polysaccharides (Kelly et al., 2016), were exclusive to BSH-positive taxa. Of particular interest, GH63’s ability to cleave α-linked L-arabinofuranosyl residues in plant hemicellulosic polysaccharides (Saito et al., 2020) may enhance microbial fitness in bile-rich environments, although its direct role in BA metabolism remains unclear. These findings highlight the metabolic versatility of BSH-positive microbes and illustrate a sophisticated interplay between carbohydrate and bile acid metabolism that supports microbial adaptation and functional dominance within the gut microbiome.

Parasite, such as helminths and protozoa, can also influence gut microbiome communities (Barash et al., 2017; McKenney et al., 2015; Aivelo and Norberg, 2018). In this study, E. bieneusi infection was associated with increased microbial diversity and elevated Bacillota_C abundance, suggesting infection-driven shifts in BA-relevant microbial taxa. Members of Bacillota are known to release immunomodulatory molecules such as peptidoglycan (Jordan et al., 2023), which may influence host immune responses. Additionally, infected individuals exhibited increased levels of BSH, 7α-HSDH, and baiB, indicating a possible upregulation of BA transformation pathways during infection. These enzymatic changes could impact lipid metabolism and immune homeostasis (Guan et al., 2022; Wang et al., 2024a). Previous research has shown that elevated cholic acid can promote the growth of Bacillota species capable of 7α-dehydroxylation (Ridlon et al., 2013). Thus, parasitic infections may enhance microbial BA metabolism, with potential consequences for host immunity and susceptibility to secondary infections (Pickard et al., 2017; Collins et al., 2023). Taken together, our results indicate that E. bieneusi infection promotes a more metabolically active and immunomodulatory gut microbiota, possibly reshaping the intestinal environment to influence host physiological status.

Metabolic profiling of BAs using advanced detection technologies offers accurate and comprehensive monitoring of their composition and concentrations, which is essential for disease prevention, diagnosis, and treatment (Liu et al., 2018). It is important to note that our findings are based on genomic potential rather than direct biochemical measurements of bile acid species. While this approach enables comprehensive and high-resolution identification of microbial metabolic capabilities (Coelho et al., 2022; Paoli et al., 2022), it does not capture actual changes in bile acid concentrations or profiles. Future studies integrating metagenomics with metabolomics and host physiological data will be critical to fully elucidate the interplay between microsporidian infection, bile acid metabolism, and host health.

This study offers a comprehensive and functional perspective on the gut microbiota of wild rodents, shedding light on the specialized role of microbial communities in BA metabolism. The dominance of Muribaculaceae, particularly CAG-485, in BA transformation highlights an underappreciated microbial function with potential implications for host metabolism. Distinct host-specific patterns in BA metabolism were observed, with wild rodents exhibiting microbial adaptations that differ markedly from those of laboratory mice and other domesticated animals—suggesting that standard model organisms may not fully capture the complexity of natural gut microbiomes. Furthermore, infection with E. bieneusi significantly altered the composition of BA-associated microbes, pointing to the dynamic responsiveness of gut microbiota to pathogen colonization. BSH-positive microbes demonstrated clear functional adaptations, including enriched glycoside hydrolase activity, supporting enhanced metabolic efficiency and survival in the bile-rich intestinal environment. These findings underscore the intricate relationship between gut microbes and host physiology, emphasizing the ecological and metabolic flexibility of the wild rodent microbiome. Future studies should aim to experimentally validate these microbial pathways and explore their influence on host health, immunity, and resistance to disease.