Mechanisms of Colonization Resistance Against Clostridioides difficile

Abstract

Clostridioides difficile is an urgent antimicrobial-resistant bacterium, causing mild to moderate and sometimes life-threatening disease. Commensal gut microbes are critical for providing colonization resistance against C difficile and can be leveraged as non-antibiotic alternative therapeutics for the prevention and treatment of C difficile infection.

Clostridioides difficile is an anaerobic, spore-forming, Gram-positive bacillus and is the leading cause of antibiotic-associated diarrhea [1]. Clostridioides difficile infection (CDI) is a significant public health problem associated with increasing morbidity, mortality, and healthcare-related costs around the globe. Clostridioides difficile is responsible for approximately 462 100 infections and 20 500 deaths per year in the United States [2]. Clostridioides difficile mediates disease through the production of 2 large toxins, TcdA and TcdB. Upon endocytosis into colonic epithelial cells, these toxins inactivate host RHO/RAC GTPases, resulting in the disruption of the actin cytoskeleton, which causes cell rounding and ultimately apoptosis or necrosis. This results in loss of cellular junctions in the colonic epithelia, significant inflammation, neutrophilic influx, and extensive tissue damage [1].

Currently, improved antibiotic stewardship and rigorous hospital infection control are the only preventative measures for CDI. Although antibiotics are the first line of treatment, they are also key risk factors in the development of CDI [3]. Current treatments for patients with CDI are the antibiotics vancomycin or fidaxomicin; however, even after successful treatment, this therapy is associated with more than 20%–25% or 10%–15%, respectively, of cases relapsing [4, 5]. In healthy individuals, the indigenous gut microbiota is essential for conferring colonization resistance against C difficile; however, antibiotic treatment disrupts this line of protection and increases susceptibility to CDI [6, 7]. For recurrent CDI, fecal microbiota transplantation (FMT) treatment has been demonstrated to cure 70%–90% of cases [8]. However, the mechanism behind FMT is not fully understood, and further investigation is needed to determine the long-term safety of this therapy.

These strategies, despite widespread implementation, have resulted in only a modest decrease in patients hospitalized for CDI, with an increase in community-associated cases [2]. The largest randomized controlled trial of probiotics has not shown a benefit in preventing CDI [9]. Therefore, there is an urgent need to identify non-antibiotic alternative therapies for treating CDI. Increased attention has been placed on identifying and understanding the intrinsic colonization resistance mechanisms used by the indigenous gut microbiota. Gut commensals have been shown to affect CDI either indirectly through the production of secondary bile acids and short-chain fatty acids (SCFAs) or directly by competing for essential nutrients or through the production of antimicrobial compounds. An ongoing effort is currently in place to exploit these mechanisms as potential treatment and therapeutic options. In this article, we discuss current and prominent literature of several microbiota-mediated mechanisms of colonization resistance against C difficile.

PERTURBANCES TO THE GUT MICROBIOTA DECREASES COLONIZATION RESISTANCE AGAINST CLOSTRIDIOIDES DIFFICILE

The gastrointestinal (GI) tract microbiota is a complex microbial ecosystem that restricts nutrients, produces antimicrobial molecules, and modulates host immunity. Stable and complex microbial communities in the gut act as an important natural barrier against enteric pathogens; therefore, alteration of the microbial composition can create a window of susceptibility for colonization and infection. Factors such as antibiotics, diet, and GI disturbances can disrupt the indigenous gut microbiota, allowing C difficile to colonize, produce toxin, and cause disease [6, 10, 11].

Antibiotic use is a primary contributing factor to CDI susceptibility. Studies on community-associated CDI reported that 60% of CDI patients had a prior exposure to antibiotics [12]. Antibiotics deplete species richness and diversity in the gut, in turn altering the metabolic environment, and ultimately creating a niche in which C difficile can thrive [6, 7, 13–15]. Schubert et al [16] demonstrated in a mouse model that concerted interactions between multiple bacterial populations are needed within the gut to establish colonization resistance against C difficile, signifying that microbial diversity is critical for preventing disease. Patients with CDI often have (1) increased levels of Lactobacillaceae and Enterobacteriaceae and (2) reduced levels of Bacteroidetes, Prevotella, and Bifidobacteria species in their stool (reviewed in [17]). Probiotic therapies geared towards restoring protective microbiota populations is a promising therapeutic option for preventing or treating CDI.

Although CDI is often referred to as a hospital- and antibiotic-associated disease, mounting evidence supports that alternative factors can also influence CDI risk [18, 19]. Metabolic interactions between the host and microbiota are instrumental for gut health, and diet can greatly impact the host metabolome and microbiota composition. The end products of complex carbohydrates metabolized by the microbiota, such as SCFAs, offer protection against CDI. Thus, Western diets high in fat and simple carbohydrates, or diets deficient in complex microbiota-accessible carbohydrates found in dietary plant polysaccharides, enhance the severity of CDI [11, 20]. Excess dietary zinc (Zn) can also perturb the gut microbiota [21]. Mice fed an excess Zn diet had significantly decreased microbiota alpha-diversity and had exacerbated epithelial damage, edema, and inflammation in a CDI mouse model.

Individuals with underlying conditions associated with an altered gut microbiota, such as inflammatory bowel disease (IBD), are at increased risk of developing CDI. Inflammatory bowel disease patients with CDI are more likely to experience recurrent infections, exacerbated clinical symptoms and increased mortality. Compared with healthy individuals, individuals with IBD have reduced microbial diversity and greater temporal instability of bacterial populations in their gut and stool samples [22]. Moreover, patients with IBD harbor an altered stool metabolome, including an increase in primary bile acids, which can promote C difficile spore germination, growth, and virulence [23]. The loss of members of the gut microbiota and increase in key metabolites are suspected to create a niche for C difficile to occupy and consequently enable expansion and disease. VanInsberghe et al [10] recently described C difficile as a “disturbance-associated opportunist,” after discovering that laxatives and diarrheal disturbances can perpetuate C difficile colonization in mice. Taken together, maintaining a diverse population of commensal bacteria in the intestine is important for maintaining colonization resistance against C difficile.

BILE ACIDS

Bile acids have a major influence on human health, microbiome composition, and disease susceptibility (reviewed in [24]). These liver-synthesized compounds are important for modulating lipoprotein, glucose, drug, and energy metabolism. Once synthesized in the liver, primary bile acids (cholate [CA] and chenodeoxycholate) travel through the small intestine, where 95% of bile acids are absorbed in the terminal ileum and enterohepatic recirculation. The small amount of bile acids that reach the large intestine are important for the metabolic functioning of the microbiota. Gut microbes encoding bile salt hydrolases (bshs) are able to deconjugate primary bile acids conjugated with a taurine or glycine [24]. A small subset of gut bacteria, including Clostridium scindens, harbor 7α-dehydroxylation activity and are able to make secondary bile acids, such as deoxycholate (DCA), lithocholate (LCA), and ursodeoxycholate (UDCA) [25]. Primary bile acids can serve as electron acceptors in fermentation, and glycine and taurine from conjugated bile acids can be used for carbon and nitrogen metabolism [26]. In addition to being essential for host and microbiota metabolism, bile acids also have antimicrobial properties. Bile acids can damage bacterial cell membranes, denature proteins, induce deoxyribonucleic acid damage, chelate iron and calcium, and mediate host innate immune responses [27].

In 2014, it was revealed that by altering the gut microbiota in mice, antibiotics ultimately changed the gut metabolic profile; specifically, the composition and concentration of bile acids [6]. The C difficile lifecycle (spore germination, growth, toxin production, and sporulation) is sensitive to different bile acids in vitro and in vivo [7, 28]. Before antibiotics, microbial-derived secondary bile acids inhibit C difficile growth. In contrast, primary bile acid levels increase after antibiotic treatment, which supports C difficile spore germination and outgrowth [6, 7, 14, 29]. In humans, patients with CDI have high concentrations of primary bile acids and undetectable levels of secondary bile acids in their stool [30]. In mice, this disruption of bile acid metabolism promotes CDI [14]. Secondary bile acid levels can be restored once the gut microbiota recovers after antibiotic treatment in mice, or by introducing donor (healthy) stool in humans via FMTs [7, 31].

Due to the strong evidence demonstrating that bile acids influence C difficile pathogenesis, manipulating bile acid pools within the gut could be a promising way to prevent or treat CDI. Bacteria that can restore secondary bile acids have been investigated for their ability to inhibit infection. A subset of species in the Clostridium genus encode a bai (bile acid inducible) operon, which allows for 7α-dehydroxylation [32–34]. Clostridium scindens was identified as a key commensal associated with partial CDI resistance through the production of the secondary bile acids, DCA and LCA, in a mouse model [35]. In a recent study, when C scindens and 3 other commensal Clostridium encoding the bai operon were grown with CA, only some were able to produce enough DCA to inhibit C difficile growth in vitro [36].

The secondary bile acid, UDCA or ursodiol, inhibits spore germination, growth, and toxin activity of C difficile in vitro [25, 28]. Ursodeoxycholate is considered a promising alternative to antibiotic treatment for CDI, and Phase 4 clinical trials are currently underway. In a CDI mouse model, UDCA pretreatment did not attenuate bacterial load in the cecum, but it did significantly reduce tissue edema, which is of great detriment to the host during CDI [37]. Ursodeoxycholate pretreatment also resulted in the increased transcript expression of 2 bile acid-activated receptors, nuclear farnesoid X receptor (FXR) and transmembrane G protein-coupled membrane receptor 5 (TGR5). Activation of FXR and TGR5 can decrease the expression of nuclear factor-κB-associated transcripts, thereby suppressing the innate immune response and inflammation [38]. This suggests UDCA may trigger a dampened innate immune response through the downstream actions of these bile acid receptors. Clostridioides difficile infection disease severity and mortality are significantly associated with inflammatory markers in both mice and humans [39], thus mediating inflammatory responses is critical for improving patient outcomes.

This paradoxical finding of UDCA causing reduced inflammation, but having no effect on bacterial burden within the gut, could also in part be explained by the findings of an additional study that revealed bile acids can interfere with TcdB activity [40]. Clostridioides difficile infection symptoms are entirely reliant on toxin production, thus inhibiting toxin activity is an attractive therapeutic option. Both primary and secondary bile acids can interact with TcdB through the CROP domain [40]. This binding induces a major conformational change within TcdB that prevents it from binding host cell receptors, which is suspected to prevent cellular internalization and intoxication. The same study also discovered that the peripheral coronary vasodilator drug, ethaverine, can mimic the same inhibition of TcdB intoxification by blocking receptor binding, and it could perhaps be administered as a potential antitoxin. Although in vivo studies are needed to validate that this approach can be used to treat CDI, this seminal finding could explain why some carriers of toxigenic C difficile lack disease symptoms.

SHORT-CHAIN FATTY ACIDS

Short-chain fatty acids are microbiota-derived metabolites produced from the fermentation of dietary fiber, of which acetate, propionate, and butyrate are the most abundant [41]. Short-chain fatty acids serve as an energy source for epithelial cells and are important for maintaining colonic health. Short-chain fatty acids can also have immunomodulatory effects, acting as an important communication system between the gut microbiota and the immune system [42].

A link between SCFAs and CDI was first discovered in a study from 1994 that found pigs fed a high-fiber diet were less susceptible to CDI [43]. The increased dietary fiber intake was suspected to support the growth of the resident gut microbes, which in turn led to increased production of acetate, propionate, and butyrate within the gut. This finding was validated in later studies demonstrating that in both human and mouse models, CDI correlates with a lower abundance of SCFA-producing bacteria and reduced SCFA concentrations in the gut [6, 44].

Recent studies have focused on deciphering the mechanistic role behind SCFA-mediated protection against CDI. Studies in mice have indicated that acetate, propionate, and butyrate all have concentration-dependent negative effects on C difficile growth and toxin production [20]. Butyrate is a major energy source for colonocytes, is critical for maintaining gut homeostasis, and was previously reported to counteract inflammatory responses in patients with ulcerative colitis. In intestinal epithelial cells (IECs), butyrate treatment attenuated toxin-mediated cellular damage and death. Mice given an oral administration of butyrate before C difficile exposure were completely protected from CDI [45]. Mice treated with butyrate after C difficile exposure had less intestinal epithelial damage and bacterial translocation compared with untreated mice. Moreover, transcript levels of hypoxia-inducible factor 1α (HIF-1α) and its target genes were elevated in response to butyrate, which are important for maintaining intestinal barrier integrity [46]. Because butyrate-mediated effects were not seen in Hif1a mutant IECs, it was proposed that butyrate stabilizes or increases epithelial cell tight junctions through the actions of HIF-1α. Although it is not clear how butyrate associates with HIF-1α, future mechanistic work may identify novel targets for drug therapies.

In addition to stabilizing the intestinal epithelial barrier during CDI, microbiota-produced SCFAs can coordinate innate immune responses. In an acute mouse model of CDI, acetate effectively attenuated C difficile expansion [47]. Acetate elicited a concerted immune signaling response in both neutrophils and interleukin (IL)C3s, triggering downstream antimicrobial and repair pathways in IECs. In neutrophils, acetate activated the free fatty acid receptor 2 (FFAR2), which induced neutrophil recruitment to the infection site and promoted IL-1β secretion. In ILC3s, acetate-induced signaling increased expression of the IL-1 receptor, which subsequently allowed for the release of IL-22 in response to IL-1β. Interleukin C3s and IL-22 have previously been shown to have a protective effect against CDI. Ragγc^−/− mice that lack ILCs had a lower percentage of survival in response to CDI than wild-type mice, although IL3Cs only had a minor contribution to resistance against CDI [48]. Il22^−/− mice challenged with C difficile carried a greater bacterial load and had more severe tissue damage than wild-type mice [49]. Interleukin-22 regulates multiple aspects of gut physiology, including the complement system and antimicrobial peptide secretion by resident microbes, to promote bacterial clearance and restore resistance against C difficile.

The discovery that acetate can influence host immunity provides a novel method for inducing these protective mechanisms. However, IL-23, a well known inducer of IL-22, is associated with increased disease severity [50]. We could speculate that IL-22 release after acetate treatment is independent of IL-23, and, instead, acetate-mediated stimulation of IL-1β is responsible for triggering IL-22 release and resulting in reduced disease severity. Crosstalk between the host immune system and commensals that translocate across the intestinal epithelial barrier was previously shown to induce IL-1β and reduce disease severity in a mouse model [51]. Although the role of acetate-producing bacteria was not investigated, this IL-1β-dependent mechanism demonstrates this molecule has an important protective advantage against CDI. Investigation using a transcriptomic approach would provide further clarification on the specific immune signaling pathways that are elicited by acetate during CDI. In addition, selective targeting of inflammatory pathways through use of microbial byproducts could be a promising therapy to alleviate CDI symptoms while avoiding exacerbation of overt inflammation.

NUTRIENT COMPETITION

Clostridioides difficile is able to fine-tune its metabolism to reflect the nutrients available in the environment; however, in the gut, C difficile must compete with the resident microbiota to acquire essential nutrients. Clostridioides difficile is auxotrophic for multiple amino acids, and it can use carbohydrates, sugar alcohols, and carboxylic acids for carbon and nitrogen metabolism. Perturbances in the diversity and richness of gut commensals could confer a great fitness advantage for C difficile, because it eliminates competing nutrient-consuming bacteria, and results in increased amino acids and carbohydrates to facilitate growth and expansion [52, 53].

Clostridioides difficile can break down components of intestinal mucin, such as sialic acid, as a source of carbon. However, C difficile lacks a sialic acid catabolism operon, and must rely on other commensals, such as Bacteroides thetaiotaomicron, for the release of sialic acid from mucosal carbohydrates [54]. Gut sialic acid levels remain low due to being readily consumed by the endogenous microbiota; however, the antibiotic-induced disruption of gut commensals results in increased intestinal sialic acid levels due to less consumption [55]. This consequently provides C difficile with access to an abundant carbon source. A C difficile mutant with compromised sialic acid metabolic activity was not able to survive within a mouse model, exemplifying the significance of this metabolite for CDI. In addition to sialic acid, gut commensals can limit the availability of succinate [56]. Succinate levels rise after use of antibiotics, in which C difficile reduces to butyrate to generate NAD⁺ to fuel the catabolism of dietary carbohydrates [55]. Targeted therapies to replenish sialic acid- and succinate-consuming commensals in CDI-patients could be of great therapeutic value for relapse prevention.

Crosstalk between the microbiota and host immune system can also influence the availability of nutrients in the gut. During CDI, SCFAs can trigger host inflammatory responses by signaling IL3Cs to release IL-22 [47]. In addition, resident gut microbes can also trigger IL-22 production [57]. Interleukin-22 transcript levels were significantly elevated in CDI mice that harbored a human-associated microbiota compared with germfree mice. Although it is not clear how the microbiota can induce IL-22 production, IL-22 was found to regulate host glycosylation and modulate the abundance of specific commensals. It is suspected that IL-22-induced glycosylation increases the amount of available carbohydrates in the lumen for gut commensals to consume. Phascolarctobacterium, a prominent consumer of succinate, was significantly more abundant in the gut in response to IL-22 signaling. This is presumably due Phascolarctobacterium’s ability to metabolize glycans produced by IL-22-mediated glycosylation. Increased carbohydrate availability promoted Phascolarctobacterium growth, leading to increased succinate consumption and limiting the availability of free succinate in the intestine for C difficile. Antibiotic-treated mice given an oral gavage of Phascolarctobacterium before challenge with C difficile had a significantly greater survival rate compared with untreated mice, demonstrating that Phascolarctobacterium could be an effective probiotic therapy for CDI.

DIRECT ANTAGONISM

Some commensal species have acquired specialized machinery for eliminating bacterial competitors in the gut. Bacterial antagonism can be exploited as a resource to search for novel agents of direct antagonism against C difficile. The commensal, Bacillus thuringiensis, produces Thuricin CD, a sactibiotic bacteriocin with narrow spectrum activity against C difficile strains [58]. Thuricin CD consists of 2 peptides, Trn-α and Trn-β, both of which are membrane-acting, forming pores in target membranes resulting in increased permeabilization, ion flux, and membrane depolarization.

To identify new probiotics that are active against C difficile, Spinler et al [59] performed a screen to identify potential candidates by testing their ability to survive in the antibiotic-treated gut. Out of a collection of tested Lactobacillus strains, Lactobacillus reuteri was the least susceptible to vancomycin, metronidazole, and fidaxomicin. In the human gut, L reuteri metabolizes glycerol into reuterin, an antimicrobial compound with broad-spectrum activity against other gut bacteria [60]. Lactobacillus reuteri significantly inhibited C difficile growth in vitro. This inhibition was dependent on reuterin production, because a reuterin-deficient L reuteri mutant was not able to impair C difficile growth. To test the effectiveness of L reuteri as a CDI therapy in vivo, glycerol and L reuteri were coadministered to clindamycin-treated mice at early stages of CDI. Codelivery significantly suppressed the C difficile burden in the gut, and it also had a minimal effect on the overall microbiota community composition indicating that L reuteri is a strong candidate for probiotic therapy.

Within the gut environment, there is a complex, concomitant production of multiple antibacterial compounds by different bacteria. This led Hanchi et al [61] to suspect that administering multiple antimicrobial agents in parallel may result in synergistic inhibitory effects. Durancin 61, a bacteriocin produced by Enterococcus durans, was tested in combination with reuterin in vitro [62]. Administration of both durancin and reuterin exhibited a more robust inhibitory effect against C difficile than either compound when used alone. Although this has not been tested in vivo, this finding suggests that combinatorial approaches to combat CDI may offer a more effective treatment option.

Combinatorial approaches using bile acids also appear to have synergistic inhibitory effects against C difficile. The bile acid 7α-dehydroxylating gut bacteria, C scindens and Clostridium sordelli, produce the tryptophan-derived antibiotics, 1-acetyl-β-carboline and turbomycin A, respectively [63]. When used in combination with the secondary bile acids, DCA and LCA, 1-acetyl-β-carboline had a synergistic inhibitory effect on C difficile growth in vitro. This study intriguingly demonstrates that certain gut microbes have acquired multiple protective mechanisms for preventing C difficile colonization.

CONCLUSIONS

Because antibiotic resistance is a prominent threat to today’s society, there is an urgent need to develop innovative approaches for combatting antibiotic-resistant bacterial infections. Recent advances in technology have greatly improved our understanding of the dynamics between C difficile, the host, and the gut microbiota. Restoration of bile acids, SCFAs, or different consortia of commensals in the gut represent promising therapies that are currently being explored. Future advances in the rational and targeted design for delivery of these metabolites to the gut are needed to effectively restore colonization resistance against C difficile.

Notes

Financial support. C. M. P. and C. M. T. are funded by the National Institute of General Medical Sciences of the National Institutes of Health under award number R35GM119438.

Supplement sponsorship. This work is part of a supplement sponsored by the National Institute of Allergy and Infectious Diseases (NIAID), part of the National Institutes of Health (NIH) and the Centers for Disease Control and Prevention (CDC).

Potential conflict of interest. C. M. T. is a scientific advisor to Locus Biosciences and a consultant for Vedanta Biosciences and Summit Therapeutics. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.