Roles of Gut Microbiota and Associated Metabolites in Clostridioides difficile Infection

Clostridioides difficile is a ubiquitous anaerobic Gram-positive spore-forming and toxin-producing bacterium. C. difficile infection (CDI), is the most common healthcare problem in the developed world (Guh et al. 2020; Khanna 2021). It is a disease primarily involving the colon of individuals whose gut microbiome has been disrupted and propagated by risk exposure factors. Alterations in the structure and function of the gut microbiome in the context of CDI create a favorable metabolic microenvironment for the life cycle of C. difficile, promoting the germination, expansion, and virulence of this important pathogen. The available approved antibiotic-based and microbiome-based medications for CDI currently have several drawbacks (McDonald et al. 2018). Antibiotics, such as vancomycin or fidaxomicin, may lead to collateral damage to the gut microbiome and fail to improve outcomes (Sehgal and Khanna 2021). Fecal microbiota transplantation, which addresses the pathophysiology of CDI, remains available in limited clinical settings as part of unclear exact mechanisms (Bednárik et al. 2025). Therefore, novel microbiome restoration therapies remain vital to curb the unmet CDI management needs.

Changes in the gut-associated microbial community composition are associated with CDI, but the mechanisms underlying this imbalance are not thoroughly understood. The main taxa within the balanced gut microbial community of human hosts are the classes Bacteroidetes (officially updated and recognized as Bacteroidia) and Clostridia (phylum Bacillota) (Tap et al. 2009). An increased abundance of Proteobacteria (officially updated and recognized as Pseudomonadota) has been proposed to characterize gut microbial imbalance (Shin et al. 2015). Proteobacteria are a minor gut-associated microbial community within a healthy host. However, a bloom of Proteobacteria is observed in patients with inflammatory bowel disease (Morgan et al. 2012), colorectal cancer (Wang et al. 2012) or necrotizing enterocolitis (Normann et al. 2013). Previous studies have also shown that reduced abundance of Bacteroidetes and Firmicutes (officially updated and recognized as Bacillota) as well as expansion of Proteobacteria were exhibited in the gut microbiota of CDI patients (Reeves et al. 2011; Mooyottu et al. 2017). A systematic search of fecal microbiota transplantation (FMT) found an increased abundance in Bacteroidetes to the detriment of Proteobacteria after fecal microbiota transplantation for CDI treatment (van Nood et al. 2013). This indicates that Proteobacteria could also serve as a metric for CDI, offering insights into an individual’s susceptibility to CDI. However, the alterations of specific members of Proteobacteria in the context of CDI are not entirely understood.

Gut microbiota can participate in critical metabolic processes of the host and shape the metabolic environment (van Prehn et al. 2021). The dominance of Bacteroidetes and Firmicutes in the human intestine ensures the production of metabolites that maintain gut homeostasis. Bacteroidetes can break down glycans and non-digestible carbohydrates for sugar harvest. Firmicutes can ferment complex carbohydrates and amino acids into short-chain fatty acids (SCFAs) (Hou et al. 2022; Gurung et al. 2024). Risk factors, such as antibiotic usage, alter gut microbiota’s structure, causing changes in amino acids, fatty acids, and bile acids and increasing susceptibility to CDI (Vliex et al. 2024). Therefore, in addition to the specific gut microbiome, metabolites could serve as the hallmark of gut microbiota dysbiosis in CDI.

Nevertheless, the results of previous studies are not always consistent. For example, in a fecal metabolome study from the 186-person cohort, 4-MPA (4-methylpentanoic acid) was identified to be elevated in patients with CDI, which was consistent with its production by C. difficile from leucine during Stickland metabolism. Noncanonical, unsaturated bile acids were also depleted in patients with CDI (Robinson et al. 2019). Another cohort study demonstrated that levels of primary bile acids, some amino acids, and fatty acids metabolites increased in feces from 30 cases of CDI compared with 25 non-CDI patients (Gu 2016). Diverse general population attributes and different analytical approaches across studies may explain these inconsistencies. Conducting studies that are less susceptible to selection bias, reverse causation, and confounding effect are critical.

We speculated that a specific metabolite might mediate the effect of Proteobacteria on CDI. Large-sample genome-wide association studies (GWAS) have identified hundreds of human single nucleotide polymorphisms (SNPs) associated with gut microbiota, facilitating the exploration of causal associations between Proteobacteria and CDI using Mendelian randomization (MR). In MR analysis, the alleles are randomly transferred from parents to offspring when the gamete is formed (Burgess et al. 2015).

Thus, in order to better characterize specific members of Proteobacteria and associated metabolite markers related to CDI susceptibility, we applied genetic instruments to assess the relations between genetically predicted gut microbiota and metabolite levels with CDI. Specifically, we applied bivariate linkage disequilibrium score regression analysis (LDSC) and MR analysis leveraging data from different populations of two ethnicities. Two-step MR extends this approach to ease mediation analysis within an MR framework. The present study aimed to investigate the detailed microbial signature of the CDI environment, especially Proteobacteria and the associated metabolic microenvironment, which will highlight therapeutic strategies targeting microbes or molecules that disrupt or enforce metabolic networks associated with CDI.

In summary, our findings suggested that Proteobacteria was genetically correlated with CDI by bivariate LDSC analysis. MR analysis indicated a suggestive genetic correlation between Sutterellaceae, Betaproteobacteria, Burkholderiales (affiliated with Proteobacteria), and CDI. Regarding a potential mechanism of metabolites, we uncovered 1,400 blood metabolites associated with the three gut microbiome taxa and CDI using two-step MR as mediation analysis. It is suggested that Burkholderiales order exerted its detrimental effects on CDI by decreasing 3-hydroxylaurate, which may provide references for the development of future interventions and potential therapeutic targets.

Previous studies have explored the association between increased Proteobacteria and CDI. A significant increase of Proteobacteria in 11 CDI patients compared with eight healthy donors enrolled in IHU-Méditerranée Infection, Marseille, France, was shown in a metagenomic analysis of gut microbiota (Amrane et al. 2019). Moreover, after fecal microbiota transplantation for recurrent CDI treatment, patients (16 patients in the infusion group) from the Academic Medical Center in Amsterdam, the Netherlands, showed an overall decrease of Proteobacteria species (Ng et al. 2020). However, these studies were performed in limited cases and in different populations. Therefore, we conducted a bivariate LDSC analysis to detect the causal relationship between gut microbiome and CDI based on 7,738 participants in the Netherlands cohort and 3,384 CDI patients. We utilized single nucleotide polymorphisms (SNPs) from GWAS summary statistics, incorporating 7,738 microbiome samples from the Netherlands and 409,432 European participants with (CDI), ensuring robust statistical power. Linkage disequilibrium score regression (LDSC) was employed to estimate genetic heritability and correlations by leveraging LD patterns from GWAS data (Bulik-Sullivan et al. 2015). First, univariate LDSC was used to assess the heritability of microbial taxa based on human genetic variation. Subsequently, bivariate LDSC quantified the genetic correlation between microbial taxa and CDI, accounting for population stratification and confounding factors that could bias GWAS estimates. This approach ensures that our findings reflect host genetic influences on microbiome composition and disease risk rather than bacterial genomic variation (Bulik-Sullivan et al. 2015). Pasteurellaceae has been reported to be positively correlated with new onset, treatment-naive Crohn’s disease (Clemente et al. 2018). Patients with ulcerative colitis and CDI contained a higher relative abundance of the genus Haemophilus than patients with ulcerative colitis only. Besides, Veillonellaceae, affiliated with Firmicutes, was also correlated with CDI, which was also positively correlated with new onset, treatment-naive Crohn’s disease (Clemente et al. 2018). The important role of Proteobacteria in CDI is associated with the disruption of the gut microbiome and proinflammation of the intestine. The deficiency in specific IgA targeting Proteobacteria is correlated with the persistence of Proteobacteria in the inflamed gut (Mirpuri et al. 2014). However, more evidence is needed to illustrate the potential mechanisms involved.

Proteobacteria is one of the most extensively studied bacterial phyla in various body sites, including the human gut and stool. Understandably, we did not identify the common species of Proteobacteria from bivariate LDSC analysis and MR analysis. MR analysis showed that intestinal taxa Sutterellaceae, Betaproteobacteria and Burkholderiales correlated positively with the incidence of CDI. In a murine model, antibiotic amoxicillin-associated enterotypes exhibited severe inflammation characterized by abundant intestinal opportunistic pathogens, including Sutterellaceae (Zhao et al. 2023). Antibiotics usage is one of the most important risk factors for the occurrence of CDI. Thus, it is rational that Sutterellaceae plays a critical detrimental role in the development of CDI. Although an elevated abundance of Sutterellaceae was observed in a study of FMT performed in 17 CDI patients, the discrepancy may be caused by limited cases (Konturek et al. 2016). Since the late 1970s, the order Burkholderiales has become prevalent in clinical settings (Hobson et al. 1995; Voronina et al. 2015). It is reported that Burkholderiales affiliated with Betaproteobacteria were highly prevalent in inflammatory bowel disease mucosa (Rudi et al. 2012). The human lysozyme milk consumption before and during enterotoxigenic Escherichia coli infection in young pigs presented decreased abundance of Burkholderiales, which was accompanied by alleviated severity of diarrhea and mitigated inflammation of intestinal mucosa (Garas et al. 2017). Although, the studies of direct correlation between Burkholderiales and CDI remains limited, we speculated that Burkholderiales affiliated with Betaproteobacteria play an important role in the diarrhea and inflammation of CDI. In addition, P. excrementihominis affiliated with Proteobacteria was negatively correlated with CDI, which exerts potential protective effect. It is demonstrated that P. excrementihominis colonizing the mice intestine up-regulated the levels of secondary bile acids, ursodeoxycholic acid (UDCA) in peripheral blood (Zhou et al. 2023). Besides, UDCA was known to be able to arrest various aspects of C. difficile life cycles in vitro and attenuate CDI related symptoms in the early stage of disease in vivo (Thanissery et al. 2017; Winston et al. 2020). The previous studies may explain the potent protective effect of P. excrementihominis on CDI.

The mediation analysis indicated that Burkholderiales exerted detrimental effects on CDI through 3-hydroxylaurate. As a predominant component in the fatty acid analysis, 3-hydroxylaurate was reported to be acylated with Leptospira interrogans lipid A. Then the complex could stimulate the production of tumor necrosis factor of mouse RAW264.7 cells (Que-Gewirth et al. 2004). The direct correlation among 3-hydroxylaurat, Burkholderiales and CDI has not been illustrated. Although two-step MR mimics the randomization process used in randomized controlled trials, the association should be confirmed in larger datasets from other genetic backgrounds. Future research in multi-omics investigations is also required to elucidate the underlying mechanism.

However, our findings have some limitations. First, genetic instrumental variables were selected reaching the threshold of p < 1 × 10⁻⁵ in order to obtain more comprehensive results, which did not meet the traditional significance standard (p < 5 × 10⁻⁸) (Cui et al. 2023). In addition, the majority of people studied were of European ancestry, which limited the generalizability of the research findings to other ethnic groups. Moreover, further subgroup analysis was impossible due to the lack of demographic data such as age, gender, and so on. Hierarchical independence control and statistical correction strategies could be applied to address taxonomic dependencies. Specifically, the integration of nonindependence in cross-species comparative analysis, such as analysis of phylogenetically independent contrasts (Felsenstein 1985), generalized least squares (Pagel 1999), phylogenetic autoregression (Gittleman and Mark 1990) and phylogenetic mixed models (Housworth et al. 2004), may help ensure hierarchical independence in Mendelian randomization analysis. We plan to incorporate these approaches in future analyses to enhance the robustness of our findings. Since Mendelian randomization is a hypothesis-driven approach (Hu et al. 2024), our results require further validation through mechanistic experiments and clinical studies to establish biological relevance.

In summary, we investigated the genetic causal effects of gut microbiota, metabolites, and CDI. Our findings revealed some significant new causal associations, including the negative effects of Burkholderiales on CDI through 3-hydroxylaurate. It may help us better understand the causal effects and identify potential therapeutic targets.