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Background. Patients with recurrent Clostridium difficile infection (CDI) have a > 60% risk of relapse, as conventional therapies do not address the underlying gastrointestinal dysbiosis. This exploratory study evaluated the safety and efficacy of bacterial spores for preventing recurrent CDI. Methods. Stool specimens from healthy donors were treated with ethanol to eliminate pathogens. The resulting spores were fractionated and encapsulated for oral delivery as SER-109. Following their response to standard-of-care antibiotics, patients in cohort 1 were treated with SER-109 on 2 consecutive days (geometric mean dose, 1.7 × 10⁹ spores), and those in cohort 2 were treated on 1 day (geometric mean dose, 1.1 × 10⁸ spores). The primary efficacy end point was absence of C. difficile-positive diarrhea during an 8-week follow-up period. Microbiome alterations were assessed. Results. Thirty patients (median age, 66.5 years; 67% female) were enrolled, and 26 (86.7%) met the primary efficacy end point. Three patients with early, self-limiting C. difficile-positive diarrhea did not require antibiotics and tested negative for C. difficile at 8 weeks; thus, 96.7% (29 of 30) achieved clinical resolution. In parallel, gut microbiota rapidly diversified, with durable engraftment of spores and no outgrowth of non-spore-forming bacteria found after SER-109 treatment. Adverse events included mild diarrhea, abdominal pain, and nausea. Conclusions. SER-109 successfully prevented CDI and had a favorable safety profile, supporting a novel microbiome-based intervention as a potential therapy for recurrent CDI.

Key Points Disease that is associated with infection by Clostridium difficile represents an urgent public health threat. The severity of C. difficile infection is determined by strain virulence, interactions with intestinal commensal microbial communities, and the host immune response to damage of the intestinal epithelium that is induced by C. difficile . The ability to sporulate and germinate is essential to C. difficile virulence. Hundreds of genes that are involved in sporulation and germination have been identified as well as a bile acid receptor that induces germination. C. difficile secretes toxin proteins that are internalized by host cells through receptor-mediated endocytosis and cause disruption to cytoskeletal architecture, which leads to cell death. Toxin-mediated cell death results in the loss of intestinal barrier integrity and the translocation of bacteria into underlying tissues. The intestinal microbiota provides colonization resistance against C. difficile infection. Commensal bacteria that are capable of converting primary bile acids to secondary bile acids inhibit the growth of C. difficile by depriving C. difficile spores of an important germinant and by increasing the concentration of secondary bile acids in the intestinal lumen, which are toxic to the vegetative form of C. difficile . Toxin-mediated damage to the epithelium activates the host inflammatory immune response. The role of the immune system is to limit epithelial damage and the dissemination of intestinal bacteria into the circulation. However, an overly robust inflammatory response can be damaging to the host and contribute to disease pathology. Treating infection with Clostridium difficile and post-antibiotic disease can be difficult. In this Review, Abt, McKenney and Pamer show how insights into spore germination, virulence and interactions with the host and microbiota can help to combat this pathogen. Clostridium difficile is a major cause of intestinal infection and diarrhoea in individuals following antibiotic treatment. Recent studies have begun to elucidate the mechanisms that induce spore formation and germination and have determined the roles of C. difficile toxins in disease pathogenesis. Exciting progress has also been made in defining the role of the microbiome, specific commensal bacterial species and host immunity in defence against infection with C. difficile . This Review will summarize the recent discoveries and developments in our understanding of C. difficile infection and pathogenesis.

Antibiotics can have significant and long-lasting effects on the gastrointestinal tract microbiota, reducing colonization resistance against pathogens including Clostridium difficile . Here we show that antibiotic treatment induces substantial changes in the gut microbial community and in the metabolome of mice susceptible to C. difficile infection. Levels of secondary bile acids, glucose, free fatty acids and dipeptides decrease, whereas those of primary bile acids and sugar alcohols increase, reflecting the modified metabolic activity of the altered gut microbiome. In vitro and ex vivo analyses demonstrate that C. difficile can exploit specific metabolites that become more abundant in the mouse gut after antibiotics, including the primary bile acid taurocholate for germination, and carbon sources such as mannitol, fructose, sorbitol, raffinose and stachyose for growth. Our results indicate that antibiotic-mediated alteration of the gut microbiome converts the global metabolic profile to one that favours C. difficile germination and growth. Antibiotics alter the intestinal microbiota and facilitate colonization of pathogens such as Clostridium difficile . Here, the authors show that antibiotic-induced shifts in the mouse gut microbiome are correlated with changes in levels of certain metabolites that C. difficile can use for germination and growth.

Predicting dynamics of host-microbial ecosystems is crucial for the rational design of bacteriotherapies. We present MDSINE, a suite of algorithms for inferring dynamical systems models from microbiome time-series data and predicting temporal behaviors. Using simulated data, we demonstrate that MDSINE significantly outperforms the existing inference method. We then show MDSINE’s utility on two new gnotobiotic mice datasets, investigating infection with Clostridium difficile and an immune-modulatory probiotic. Using these datasets, we demonstrate new capabilities, including accurate forecasting of microbial dynamics, prediction of stable sub-communities that inhibit pathogen growth, and identification of bacteria most crucial to community integrity in response to perturbations.

Fecal microbiota transplantation (FMT) is a highly effective therapy for recurrent Clostridium difficile infection (R-CDI), but its mechanisms remain poorly understood. Emerging evidence suggests that gut bile acids have significant influence on the physiology of C. difficile, and therefore on patient susceptibility to recurrent infection. We analyzed spore germination of 10 clinical C. difficile isolates exposed to combinations of bile acids present in patient feces before and after FMT. Bile acids at concentrations found in patients' feces prior to FMT induced germination of C. difficile, although with variable potency across different strains. However, bile acids at concentrations found in patients after FMT did not induce germination and inhibited vegetative growth of all C. difficile strains. Sequencing of the newly identified germinant receptor in C. difficile, CspC, revealed a possible correspondence of variation in germination responses across isolates with mutations in this receptor. This may be related to interstrain variability in spore germination and vegetative growth in response to bile acids seen in this and other studies. These results support the idea that intra-colonic bile acids play a key mechanistic role in the success of FMT, and suggests that novel therapeutic alternatives for treatment of R-CDI may be developed by targeted manipulation of bile acid composition in the colon.

Clostridium difficile is the primary cause of nosocomial diarrhea and pseudomembranous colitis. It produces dormant spores, which serve as an infectious vehicle responsible for transmission of the disease and persistence of the organism in the environment. In Bacillus subtilis, the sin locus coding SinR (113 aa) and SinI (57 aa) is responsible for sporulation inhibition. In B. subtilis, SinR mainly acts as a repressor of its target genes to control sporulation, biofilm formation, and autolysis. SinI is an inhibitor of SinR, so their interaction determines whether SinR can inhibit its target gene expression. The C. difficile genome carries two sinR homologs in the operon that we named sinR and sinR', coding for SinR (112 aa) and SinR' (105 aa), respectively. In this study, we constructed and characterized sin locus mutants in two different C. difficile strains R20291 and JIR8094, to decipher the locus's role in C. difficile physiology. Transcriptome analysis of the sinRR' mutants revealed their pleiotropic roles in controlling several pathways including sporulation, toxin production, and motility in C. difficile. Through various genetic and biochemical experiments, we have shown that SinR can regulate transcription of key regulators in these pathways, which includes sigD, spo0A, and codY. We have found that SinR' acts as an antagonist to SinR by blocking its repressor activity. Using a hamster model, we have also demonstrated that the sin locus is needed for successful C. difficile infection. This study reveals the sin locus as a central link that connects the gene regulatory networks of sporulation, toxin production, and motility; three key pathways that are important for C. difficile pathogenesis.

Perturbations to the gut microbiota can result in a loss of colonization resistance against gastrointestinal pathogens such as Clostridium difficile . Although C. difficile infection is commonly associated with antibiotic use, the precise alterations to the microbiota associated with this loss in function are unknown. We used a variety of antibiotic perturbations to generate a diverse array of gut microbiota structures, which were then challenged with C. difficile spores. Across these treatments we observed that C. difficile resistance was never attributable to a single organism, but rather it was the result of multiple microbiota members interacting in a context-dependent manner. Using relative abundance data, we built a machine learning regression model to predict the levels of C. difficile that were found 24 h after challenging the perturbed communities. This model was able to explain 77.2% of the variation in the observed number of C. difficile per gram of feces. This model revealed important bacterial populations within the microbiota, which correlation analysis alone did not detect. Specifically, we observed that populations associated with the Porphyromonadaceae , Lachnospiraceae , Lactobacillus , and Alistipes were protective and populations associated with Escherichia and Streptococcus were associated with high levels of colonization. In addition, a population affiliated with the Akkermansia indicated a strong context dependency on other members of the microbiota. Together, these results indicate that individual bacterial populations do not drive colonization resistance to C. difficile . Rather, multiple diverse assemblages act in concert to mediate colonization resistance. IMPORTANCE The gastrointestinal tract harbors a complex community of bacteria, known as the microbiota, which plays an integral role preventing its colonization by gut pathogens. This resistance has been shown to be crucial for protection against Clostridium difficile infections (CDI), which are the leading source of hospital-acquired infections in the United States. Antibiotics are a major risk factor for acquiring CDI due to their effect on the normal structure of the indigenous gut microbiota. We found that diverse antibiotic perturbations gave rise to altered communities that varied in their susceptibility to C. difficile colonization. We found that multiple coexisting populations, not one specific population of bacteria, conferred resistance. By understanding the relationships between C. difficile and members of the microbiota, it will be possible to better manage this important infection. The gastrointestinal tract harbors a complex community of bacteria, known as the microbiota, which plays an integral role preventing its colonization by gut pathogens. This resistance has been shown to be crucial for protection against Clostridium difficile infections (CDI), which are the leading source of hospital-acquired infections in the United States. Antibiotics are a major risk factor for acquiring CDI due to their effect on the normal structure of the indigenous gut microbiota. We found that diverse antibiotic perturbations gave rise to altered communities that varied in their susceptibility to C. difficile colonization. We found that multiple coexisting populations, not one specific population of bacteria, conferred resistance. By understanding the relationships between C. difficile and members of the microbiota, it will be possible to better manage this important infection.

Antimicrobial therapy plays a central role in the pathogenesis of Clostridium difficile infection (CDI), presumably through disruption of indigenous intestinal microflora, thereby allowing C. difficile to grow and produce toxin. Investigations involving animal models and studies performed in vitro suggest that inhibitory activity against C. difficile and differences in the propensity to stimulate toxin production may also influence the likelihood that particular drugs may cause CDI. Although nearly all antimicrobial classes have been associated with CDI, clindamycin, third-generation cephalosporins, and penicillins have traditionally been considered to harbor the greatest risk. Recent studies have also implicated fluoroquinolones as high-risk agents, a finding that is most likely to be related in part to increasing fluoroquinolone resistance among epidemic strains (i.e., restriction-endonuclease analysis group BI/North American PFGE type 1 strains) and some nonepidemic strains of C. difficile. Restrictions in the use of clindamycin and third-generation cephalosporins have been associated with reductions in CDI. Because use of any antimicrobial has the potential to induce the onset of CDI and disease caused by other health care–associated pathogens, antimicrobial stewardship programs that promote judicious use of antimicrobials are encouraged in concert with environmental and infection control–related efforts.

Fidaxomicin (FDX) is a novel antimicrobial agent with narrow-spectrum and potent bactericidal activity against Clostridium difficile. In recent clinical trials, FDX was superior to vancomycin in preventing recurrences of C. difficile infection. A possible mechanism of reducing recurrence may be through an inhibitory effect on sporulation. The effect of FDX and its major metabolite, OP-1118, on C. difficile growth and sporulation kinetics was compared with that of vancomycin, metronidazole, and rifaximin. Drugs at subminimum inhibitory concentrations (sub-MICs) were added to cells at an early stationary phase of growth; this was followed by collection of cells at various intervals for quantitation of total viable cell and heat-resistant spore counts on taurocholate-containing media. The effect of the drugs at 2—2.5× MIC on the expression of sporulation genes in C. difficile was also compared using quantitative reverse-transcriptase polymerase chain reaction. Both FDX and OP-1118 (1/4× MIC) inhibited sporulation when added to early-stationary-phase cells in C. difficile strains, including the epidemic NAP1/BI/027 strain. In contrast, vancomycin, metronidazole, and rifaximin (at similar sub-MICs) did not inhibit sporulation. The number of spores following treatment with comparator drugs increased to the same level as the no-drug control treatment. Expression of mother cell—specific (spoIIID) and forespore-specific (spoIIR) sporulation genes also was inhibited by FDX and OP-1118 but not significantly by vancomycin. Both FDX and OP-1118 (unlike vancomycin, rifaximin, and metronidazole) effectively inhibited sporulation by C. difficile. The inhibitory effect of FDX on C. difficile sporulation may contribute to its superior performance in sustaining clinical response and reducing recurrences and may also be beneficial in decreasing shedding and transmission of this pathogen.

Clostridium difficile (C. difficile) is an anaerobic gram-positive pathogen that is the leading cause of nosocomial bacterial infection globally. C. difficile infection (CDI) typically occurs after ingestion of infectious spores by a patient that has been treated with broad-spectrum antibiotics. While CDI is a toxin-mediated disease, transmission and pathogenesis are dependent on the ability to produce viable spores. These spores must become metabolically active (germinate) in order to cause disease. C. difficile spore germination occurs when spores encounter bile salts and other co-germinants within the small intestine, however, the germination signaling cascade is unclear. Here we describe a signaling role for Ca2+ during C. difficile spore germination and provide direct evidence that intestinal Ca2+ coordinates with bile salts to stimulate germination. Endogenous Ca2+ (released from within the spore) and a putative AAA+ ATPase, encoded by Cd630_32980, are both essential for taurocholate-glycine induced germination in the absence of exogenous Ca2+. However, environmental Ca2+ replaces glycine as a co-germinant and circumvents the need for endogenous Ca2+ fluxes. Cd630_32980 is dispensable for colonization in a murine model of C. difficile infection and ex vivo germination in mouse ileal contents. Calcium-depletion of the ileal contents prevented mutant spore germination and reduced WT spore germination by 90%, indicating that Ca2+ present within the gastrointestinal tract plays a critical role in C. difficile germination, colonization, and pathogenesis. These data provide a biological mechanism that may explain why individuals with inefficient intestinal calcium absorption (e.g., vitamin D deficiency, proton pump inhibitor use) are more prone to CDI and suggest that modulating free intestinal calcium is a potential strategy to curb the incidence of CDI.