The design, synthesis, and inhibition of Clostridioides difficile spore germination by acyclic and bicyclic tertiary amide analogs of cholate.

Clostridioides difficile infection (CDI) is a major identifiable cause of antibiotic-associated diarrhea. In our previous study (J. Med. Chem., 2018, 61, 6759-6778), we have identified N-phenyl-cholan-24-amide as a potent inhibitor of spore germination. The most potent compounds in our previous work are N-arylamides. We were interested in the role that the conformation of the amide plays in activity. Previous research has shown that secondary N-arylamides exist exclusively in the coplanar trans conformation while tertiary N-methyl-N-arylamides exist in a non-planar, cis conformation. The N-methyl-N-phenyl-cholan-24-amide was 17-fold less active compared to the parent compounds suggesting the importance of the orientation of the phenyl ring. To lock the phenyl ring into a trans conformation, cyclic tertiary amides were prepared. Indoline and quinoline cholan-24-amides were both inhibitors of spore germination; however, the indoline analogs were most potent. Isoindoline and isoquinoline amides were inactive. We found that the simple indoline derivative gave an IC50 value of 1 µM, while the 5'-fluoro-substituted compound (5d) possessed an IC50 of 400 nM. To our knowledge, 5d is the most potent known spore germination inhibitor described to date. Taken together, our results indicate that the trans, coplanar conformation of the phenyl ring is required for potent inhibition.

Comparison of sporulation and germination conditions for Clostridium perfringens type A and G strains.

Clostridium perfringens is a spore forming, anaerobic, Gram-positive bacterium that causes a range of diseases in humans and animals. C. perfringens forms spores, structures that are derived from the vegetative cell under conditions of nutrient deprivation and that allows survival under harsh environmental conditions. To return to vegetative growth, C. perfringens spores must germinate when conditions are favorable. Previous work in analyzing C. perfringens spore germination has produced strain-specific results. Hence, we analyzed the requirements for spore formation and germination in seven different C. perfringens strains. Our data showed that C. perfringens sporulation conditions are strain-specific, but germination responses are homogenous in all strains tested. C. perfringens spores can germinate using two distinct pathways. The first germination pathway (the amino acid-only pathway or AA) requires L-alanine, L-phenylalanine, and sodium ions (Na+) as co-germinants. L-arginine is not a required germinant but potentiates germination. The AA pathway is inhibited by aromatic amino acids and potassium ions (K+). Bicarbonate (HCO3-), on the other hand, bypasses potassium-mediated inhibition of C. perfringens spore germination through the AA pathway. The second germination pathway (the bile salt / amino acid pathway or BA) is more promiscuous and is activated by several bile salts and amino acids. In contrast to the AA pathway, the BA pathway is insensitive to Na+, although it can be activated by either K+ or HCO3-. We hypothesize that some C. perfringens strains may have evolved these two distinct germination pathways to ensure spore response to different host environments.

Mechanism of germination inhibition of Clostridioides difficile spores by an aniline substituted cholate derivative (CaPA).

Clostridioides difficile infection (CDI) is the major identifiable cause of antibiotic-associated diarrhea and has been declared an urgent threat by the CDC. C. difficile forms dormant and resistant spores that serve as infectious vehicles for CDI. To cause disease, C. difficile spores recognize taurocholate and glycine to trigger the germination process. In contrast to other sporulating bacteria, C. difficile spores are postulated to use a protease complex, CspABC, to recognize its germinants. Since spore germination is required for infection, we have developed anti-germination approaches for CDI prophylaxis. Previously, the bile salt analog CaPA (an aniline-substituted cholic acid) was shown to block spore germination and protect rodents from CDI caused by multiple C. difficile strains and isolates. In this study, we found that CaPA is an alternative substrate inhibitor of C. difficile spore germination. By competing with taurocholate for binding, CaPA delays C. difficile spore germination and reduces spore viability, thus diminishing the number of outgrowing vegetative bacteria. We hypothesize that the reduction of toxin-producing bacterial burden explains CaPA's protective activity against murine CDI. Previous data combined with our results suggests that CaPA binds tightly to C. difficile spores in a CspC-dependent manner and irreversibly traps spores in an alternative, time-delayed, and low yield germination pathway. Our results are also consistent with kinetic data suggesting the existence of at least two distinct bile salt binding sites in C. difficile spores.

A High-Carbohydrate Diet Prolongs Dysbiosis and Clostridioides difficile Carriage and Increases Delayed Mortality in a Hamster Model of Infection.

Studies using mouse models of Clostridioides difficile infection (CDI) have demonstrated a variety of relationships between dietary macronutrients on antibiotic-associated CDI; however, few of these effects have been examined in more susceptible hamster models of CDI. In this study, we investigated the effect of a high-carbohydrate diet previously shown to protect mice from CDI on the progression and resolution of CDI in a hamster disease model, with 10 animals per group. Hamsters fed the high-carbohydrate diet developed distinct diet-specific microbiomes during antibiotic treatment and CDI, with lower diversity, persistent C. difficile carriage, and delayed microbiome restoration. In contrast to CDI protection in mice, most hamsters fed a high-carbohydrate diet developed fulminant CDI including several cases of late-onset CDI, that were not observed in hamsters fed a standard lab diet. We speculate that prolonged high-carbohydrate diet-specific dysbiosis in these animals allowed C. difficile to persist in the gut of the animals where they could proliferate postvancomycin treatment, leading to delayed CDI onset. This study, along with similar studies in mouse models of CDI, suggests some high-carbohydrate diets may promote antibiotic-associated dysbiosis and long-term C. difficile carriage, which may later convert to symptomatic CDI. IMPORTANCE The effects of diet on CDI are not completely known. Here, we used a high-carbohydrate diet previously shown to protect mice against CDI to assess its effect on a hamster model of CDI and paradoxically found that it promoted dysbiosis, C. difficile carriage, and higher mortality. A common thread in both mouse and hamster experimental models was that the high-carbohydrate diet promoted dysbiosis and long-term carriage of C. difficile, which may have converted to fulminant CDI only in the highly susceptible hamster model system. If diets high in carbohydrates also promote dysbiosis and C. difficile carriage in humans, then these diets might paradoxically increase chances of CDI relapse despite their protective effects against primary CDI.

An Aniline-Substituted Bile Salt Analog Protects both Mice and Hamsters from Multiple Clostridioides difficile Strains.

Clostridioides difficile infection (CDI) is the major identifiable cause of antibiotic-associated diarrhea. The emergence of hypervirulent C. difficile strains has led to increases in both hospital- and community-acquired CDI. Furthermore, the rate of CDI relapse from hypervirulent strains can reach up to 25%. Thus, standard treatments are rendered less effective, making new methods of prevention and treatment more critical. Previously, the bile salt analog CamSA (cholic acid substituted with m-aminosulfonic acid) was shown to inhibit spore germination in vitro and protect mice and hamsters from C. difficile strain 630. Here, we show that CamSA was less active in preventing spore germination by other C. difficile ribotypes, including the hypervirulent strain R20291. The strain-specific in vitro germination activity of CamSA correlated with its ability to prevent CDI in mice. Additional bile salt analogs were screened for in vitro germination inhibition activity against strain R20291, and the most active compounds were tested against other strains. An aniline-substituted bile salt analog, CaPA (cholic acid substituted with phenylamine), was found to be a better antigerminant than CamSA against eight different C. difficile strains. In addition, CaPA was capable of reducing, delaying, or preventing murine CDI signs with all strains tested. CaPA-treated mice showed no obvious toxicity and showed minor effects on their gut microbiome. CaPA's efficacy was further confirmed by its ability to prevent CDI in hamsters infected with strain 630. These data suggest that C. difficile spores respond to germination inhibitors in a strain-dependent manner. However, careful screening can identify antigerminants with broad CDI prophylaxis activity.

Studies on the Importance of the 7α-, and 12α- hydroxyl groups of N-Aryl-3α,7α,12α-trihydroxy-5ß-cholan-24-amides on their Antigermination Activity Against a Hypervirulent Strain of Clostridioides (Clostridium) difficile.

Chenodeoxycholic acid (CDCA) is a natural germination inhibitor for C. difficile spores. In our previous study (J. Med. Chem., 2018, 61, 6759-6778), we identified N-phenyl-3α,7α,12α-trihydroxy-5ß-cholan-24-amide as an inhibitor of C. difficile strain R20291 with an IC50 of 1.8 µM. Studies of bile salts on spore germination have shown that chenodeoxycholate, ursodeoxycholate and lithocholate are more potent inhibitors of germination compared to cholate. Given this, we created amide analogs of chenodeoxycholic, deoxycholic, lithocholic and ursodeoxycholic acids using amines identified from our previous studies. We found that chenodeoxy- and deoxycholate derivatives were active with potencies equivalent to those for cholanamides. This indicates that only 2 out of the 3 hydroxyl groups are needed for activity and that the alpha stereochemistry at position 7 is required for inhibition of spore germination.

Pharmacokinetics of CamSA, a potential prophylactic compound against Clostridioides difficile infections.

Clostridioides difficile infections (CDI) are the leading cause of nosocomial antibiotic-associated diarrhea. C. difficile produces dormant spores that serve as infectious agents. Bile salts in the gastrointestinal tract signal spores to germinate into toxin-producing cells. As spore germination is required for CDI onset, anti-germination compounds may serve as prophylactics. CamSA, a synthetic bile salt, was previously shown to inhibit C. difficile spore germination in vitro and in vivo. Unexpectedly, a single dose of CamSA was sufficient to offer multi-day protection from CDI in mice without any observable toxicity. To study this intriguing protection pattern, we examined the pharmacokinetic parameters of CamSA. CamSA was stable to the gut of antibiotic-treated mice but was extensively degraded by the microbiota of non-antibiotic-treated animals. Our data also suggest that CamSA's systemic absorption is minimal since it is retained primarily in the intestinal lumen and liver. CamSA shows weak interactions with CYP3A4, a P450 hepatic isozyme involved in drug metabolism and bile salt modification. Like other bile salts, CamSA seems to undergo enterohepatic circulation. We hypothesize that the cycling of CamSA between the liver and intestines serves as a slow-release mechanism that allows CamSA to be retained in the gastrointestinal tract for days. This model explains how a single CamSA dose can prevent murine CDI even though spores are present in the animal's intestine for up to four days post-challenge.

A High-Fat/High-Protein, Atkins-Type Diet Exacerbates Clostridioides (Clostridium) difficile Infection in Mice, whereas a High-Carbohydrate Diet Protects.

Clostridioides difficile (formerly Clostridium difficile) infection (CDI) can result from the disruption of the resident gut microbiota. Western diets and popular weight-loss diets drive large changes in the gut microbiome; however, the literature is conflicted with regard to the effect of diet on CDI. Using the hypervirulent strain C. difficile R20291 (RT027) in a mouse model of antibiotic-induced CDI, we assessed disease outcome and microbial community dynamics in mice fed two high-fat diets in comparison with a high-carbohydrate diet and a standard rodent diet. The two high-fat diets exacerbated CDI, with a high-fat/high-protein, Atkins-like diet leading to severe CDI and 100% mortality and a high-fat/low-protein, medium-chain-triglyceride (MCT)-like diet inducing highly variable CDI outcomes. In contrast, mice fed a high-carbohydrate diet were protected from CDI, despite the high levels of refined carbohydrate and low levels of fiber in the diet. A total of 28 members of the Lachnospiraceae and Ruminococcaceae decreased in abundance due to diet and/or antibiotic treatment; these organisms may compete with C. difficile for amino acids and protect healthy animals from CDI in the absence of antibiotics. Together, these data suggest that antibiotic treatment might lead to loss of C. difficile competitors and create a favorable environment for C. difficile proliferation and virulence with effects that are intensified by high-fat/high-protein diets; in contrast, high-carbohydrate diets might be protective regardless of the source of carbohydrate or of antibiotic-driven loss of C. difficile competitors.IMPORTANCE The role of Western and weight-loss diets with extreme macronutrient composition in the risk and progression of CDI is poorly understood. In a longitudinal study, we showed that a high-fat/high-protein, Atkins-type diet greatly exacerbated antibiotic-induced CDI, whereas a high-carbohydrate diet protected, despite the high monosaccharide and starch content. Our study results, therefore, suggest that popular high-fat/high-protein weight-loss diets may enhance CDI risk during antibiotic treatment, possibly due to the synergistic effects of a loss of the microorganisms that normally inhibit C. difficile overgrowth and an abundance of amino acids that promote C. difficile overgrowth. In contrast, a high-carbohydrate diet might be protective, despite reports on the recent evolution of enhanced carbohydrate metabolism in C. difficile.

Effect of the Synthetic Bile Salt Analog CamSA on the Hamster Model of Clostridium difficile Infection.

Clostridium difficile infection (CDI) is the leading cause of antibiotic-associated diarrhea and has gained worldwide notoriety due to emerging hypervirulent strains and the high incidence of recurrence. We previously reported protection of mice from CDI using the antigerminant bile salt analog CamSA. Here we describe the effects of CamSA in the hamster model of CDI. CamSA treatment of hamsters showed no toxicity and did not affect the richness or diversity of gut microbiota; however, minor changes in community composition were observed. Treatment of C. difficile-challenged hamsters with CamSA doubled the mean time to death, compared to control hamsters. However, CamSA alone was insufficient to prevent CDI in hamsters. CamSA in conjunction with suboptimal concentrations of vancomycin led to complete protection from CDI in 70% of animals. Protected animals remained disease-free at least 30 days postchallenge and showed no signs of colonic tissue damage. In a delayed-treatment model of hamster CDI, CamSA was unable to prevent infection signs and death. These data support a putative model in which CamSA reduces the number of germinating C. difficile spores but does not keep all of the spores from germinating. Vancomycin halts division of any vegetative cells that are able to grow from spores that escape CamSA.

The Design, Synthesis, and Characterizations of Spore Germination Inhibitors Effective against an Epidemic Strain of Clostridium difficile.

Clostridium difficile infections (CDI), particularly those caused by the BI/NAP1/027 epidemic strains, are challenging to treat. One method to address this disease is to prevent the development of CDI by inhibiting the germination of C. difficile spores. Previous studies have identified cholic amide m-sulfonic acid, CamSA, as an inhibitor of spore germination. However, CamSA is inactive against the hypervirulent strain R20291. To circumvent this problem, a series of cholic acid amides were synthesized and tested against R20291. The best compound in the series was the simple phenyl amide analogue which possessed an IC50 value of 1.8 µM, more than 225 times as potent as the natural germination inhibitor, chenodeoxycholate. This is the most potent inhibitor of C. difficile spore germination described to date. QSAR and molecular modeling analysis demonstrated that increases in hydrophobicity and decreases in partial charge or polar surface area were correlated with increases in potency.

Inhibitory effect of indole analogs against Paenibacillus larvae, the causal agent of American foulbrood disease.

Paenibacillus larvae, a Gram-positive bacterium, causes American foulbrood (AFB) in honey bee larvae (Apis mellifera Linnaeus [Hymenoptera: Apidae]). P. larvae spores exit dormancy in the gut of bee larvae, the germinated cells proliferate, and ultimately bacteremia kills the host. Hence, spore germination is a required step for establishing AFB disease. We previously found that P. larvae spores germinate in response to l-tyrosine plus uric acid in vitro. Additionally, we determined that indole and phenol blocked spore germination. In this work, we evaluated the antagonistic effect of 35 indole and phenol analogs and identified strong inhibitors of P. larvae spore germination in vitro. We further tested the most promising candidate, 5-chloroindole, and found that it significantly reduced bacterial proliferation. Finally, feeding artificial worker jelly containing anti-germination compounds to AFB-exposed larvae significantly decreased AFB infection in laboratory-reared honey bee larvae. Together, these results suggest that inhibitors of P. larvae spore germination could provide another method to control AFB.

ent-Labdane Diterpenoids from the Aerial Parts of Eupatorium obtusissmum.

Six new ent-labdane diterpenoids, uasdlabdanes A-F (1-6), were isolated from the aerial parts of Eupatorium obtusissmum. The new structures were elucidated through spectroscopic and spectrometric data analyses. The absolute configurations of compounds 1 and 2 were established by X-ray crystallography, and those of 3-6, by comparison of experimental and calculated electronic circular dichroism spectra. The antiproliferative activity of the compounds was studied in a panel of six representative human solid tumor cell lines and showed GI50 values ranging from 19 to >100 µM.

Comparison of in vitro methods for the production of Paenibacillus larvae endospores.

Paenibacillus larvae endospores are the infectious particles of the honey bee brood disease, American Foulbrood. We demonstrate that our previously published protocol (Alvarado et al., 2013) consistently yields higher numbers and purer preparations of P. larvae endospores, than previously described protocols, regardless of the strain tested (B-3650, B-3554 or B-3685).

How enteric pathogens know they hit the sweet spot.

EVALUATION OF: Ng KM, Ferreyra JA, Higginbottom SK et al. Microbiota-liberated host sugars facilitate post-antibiotic expansion of enteric pathogens. Nature 502(7469), 96-99 (2013). The human gut microbiota is a complex system of commensal microorganisms required for normal host physiology. Disruption of this protective barrier by antibiotics creates opportunities for enteric pathogens to establish infections. Although the correlation between the use of antibiotics and enteric infections have been known for some time, the specific signals that allow enteric pathogens to recognize a susceptible host have not been determined. In a recent article, Ng et al. demonstrated that the expansion of both Salmonella typhimurium and Clostridium difficile infections is enhanced by the availability of host-specific sugars liberated from the intestinal mucus by commensal bacteria. These results show how antibiotic removal of specific species from the gut microbiome allows symbiotic functions to be hijacked by pathogenic species.

Fate of ingested Clostridium difficile spores in mice.

Clostridium difficile infection (CDI) is a leading cause of antibiotic-associated diarrhea, a major nosocomial complication. The infective form of C. difficile is the spore, a dormant and resistant structure that forms under stress. Although spore germination is the first committed step in CDI onset, the temporal and spatial distribution of ingested C. difficile spores is not clearly understood. We recently reported that CamSA, a synthetic bile salt analog, inhibits C. difficile spore germination in vitro and in vivo. In this study, we took advantage of the anti-germination activity of bile salts to determine the fate of ingested C. difficile spores. We tested four different bile salts for efficacy in preventing CDI. Since CamSA was the only anti-germinant tested able to prevent signs of CDI, we characterized CamSa's in vitro stability, distribution, and cytotoxicity. We report that CamSA is stable to simulated gastrointestinal (GI) environments, but will be degraded by members of the natural microbiota found in a healthy gut. Our data suggest that CamSA will not be systemically available, but instead will be localized to the GI tract. Since in vitro pharmacological parameters were acceptable, CamSA was used to probe the mouse model of CDI. By varying the timing of CamSA dosage, we estimated that C. difficile spores germinated and established infection less than 10 hours after ingestion. We also showed that ingested C. difficile spores rapidly transited through the GI tract and accumulated in the colon and cecum of CamSA-treated mice. From there, C. difficile spores were slowly shed over a 96-hour period. To our knowledge, this is the first report of using molecular probes to obtain disease progression information for C. difficile infection.

A new strategy for the prevention of Clostridium difficile infection.

BACKGROUND: Clostridium difficile infection (CDI) is a leading cause of antibiotic-associated diarrhea. The infective form of C. difficile is the spore, but the vegetative bacterium causes the disease. Because C. difficile spore germination is required for symptomatic infection, antigermination approaches could lead to the prevention of CDI. We recently reported that CamSA, a bile salt analog, inhibits C. difficile spore germination in vitro. METHODS: Mice infected with massive inocula of C. difficile spores were treated with different concentrations of CamSA and monitored for CDI signs. C. difficile spore and vegetative cells were counted in feces from infected mice. RESULTS: A single 50-mg/kg dose of CamSA prevented CDI in mice without any observable toxicity. Lower CamSA doses resulted in delayed CDI onset and less severe signs of disease. Ingested C. difficile spores were quantitatively recovered from feces of CamSA-protected mice. CONCLUSIONS: Our results support a mechanism whereby the antigermination effect of CamSA is responsible for preventing CDI signs. This approach represents a new paradigm in CDI treatment. Instead of further compromising the microbiota of CDI patients with strong antibiotics, antigermination therapy could serve as a microbiota surrogate to curtail C. difficile colonization of antibiotic-treated patients.

Requirements for in vitro germination of Paenibacillus larvae spores.

Paenibacillus larvae is the causative agent of American foulbrood (AFB), a disease affecting honey bee larvae. First- and second-instar larvae become infected when they ingest food contaminated with P. larvae spores. The spores then germinate into vegetative cells that proliferate in the midgut of the honey bee. Although AFB affects honey bees only in the larval stage, P. larvae spores can be distributed throughout the hive. Because spore germination is critical for AFB establishment, we analyzed the requirements for P. larvae spore germination in vitro. We found that P. larvae spores germinated only in response to l-tyrosine plus uric acid under physiologic pH and temperature conditions. This suggests that the simultaneous presence of these signals is necessary for spore germination in vivo. Furthermore, the germination profiles of environmentally derived spores were identical to those of spores from a biochemically typed strain. Because l-tyrosine and uric acid are the only required germinants in vitro, we screened amino acid and purine analogs for their ability to act as antagonists of P. larvae spore germination. Indole and phenol, the side chains of tyrosine and tryptophan, strongly inhibited P. larvae spore germination. Methylation of the N-1 (but not the C-3) position of indole eliminated its ability to inhibit germination. Identification of the activators and inhibitors of P. larvae spore germination provides a basis for developing new tools to control AFB.

Cooperativity and interference of germination pathways in Bacillus anthracis spores.

Spore germination is the first step to Bacillus anthracis pathogenicity. Previous work has shown that B. anthracis spores use germination (Ger) receptors to recognize amino acids and nucleosides as germinants. Genetic analysis has putatively paired each individual Ger receptor with a specific germinant. However, Ger receptors seem to be able to partially compensate for each other and recognize alternative germinants. Using kinetic analysis of B. anthracis spores germinated with inosine and L-alanine, we previously determined kinetic parameters for this germination process and showed binding synergy between the cogerminants. In this work, we expanded our kinetic analysis to determine kinetic parameters and binding order for every B. anthracis spore germinant pair. Our results show that germinant binding can exhibit positive, neutral, or negative cooperativity. Furthermore, different germinants can bind spores by either a random or an ordered mechanism. Finally, simultaneous triggering of multiple germination pathways shows that germinants can either cooperate or interfere with each other during the spore germination process. We postulate that the complexity of germination responses may allow B. anthracis spores to respond to different environments by activating different germination pathways.

Progesterone analogs influence germination of Clostridium sordellii and Clostridium difficile spores in vitro.

Clostridium sordellii and Clostridium difficile are closely related anaerobic Gram-positive, spore-forming human pathogens. C. sordellii and C. difficile form spores that are believed to be the infectious form of these bacteria. These spores return to toxin-producing vegetative cells upon binding to small molecule germinants. The endogenous compounds that regulate clostridial spore germination are not fully understood. While C. sordellii spores require three structurally distinct amino acids to germinate, the occurrence of postpregnancy C. sordellii infections suggests that steroidal sex hormones might regulate its capacity to germinate. On the other hand, C. difficile spores require taurocholate (a bile salt) and glycine (an amino acid) to germinate. Bile salts and steroid hormones are biosynthesized from cholesterol, suggesting that the common sterane structure can affect the germination of both C. sordellii and C. difficile spores. Therefore, we tested the effect of sterane compounds on C. sordellii and C. difficile spore germination. Our results show that both steroid hormones and bile salts are able to increase C. sordellii spore germination rates. In contrast, a subset of steroid hormones acted as competitive inhibitors of C. difficile spore germination. Thus, even though C. sordellii and C. difficile are phylogenetically related, the two species' spores respond differently to steroidal compounds.

Mapping interactions between germinants and Clostridium difficile spores.

Germination of Clostridium difficile spores is the first required step in establishing C. difficile-associated disease (CDAD). Taurocholate (a bile salt) and glycine (an amino acid) have been shown to be important germinants of C. difficile spores. In the present study, we tested a series of glycine and taurocholate analogs for the ability to induce or inhibit C. difficile spore germination. Testing of glycine analogs revealed that both the carboxy and amino groups are important epitopes for recognition and that the glycine binding site can accommodate compounds with more widely separated termini. The C. difficile germination machinery also recognizes other hydrophobic amino acids. In general, linear alkyl side chains are better activators of spore germination than their branched analogs. However, L-phenylalanine and L-arginine are also good germinants and are probably recognized by distinct binding sites. Testing of taurocholate analogs revealed that the 12-hydroxyl group of taurocholate is necessary, but not sufficient, to activate spore germination. In contrast, the 6- and 7-hydroxyl groups are required for inhibition of C. difficile spore germination. Similarly, C. difficile spores are able to detect taurocholate analogs with shorter, but not longer, alkyl amino sulfonic acid side chains. Furthermore, the sulfonic acid group can be partially substituted with other acidic groups. Finally, a taurocholate analog with an m-aminobenzenesulfonic acid side chain is a strong inhibitor of C. difficile spore germination. In conclusion, C. difficile spores recognize both amino acids and taurocholate through multiple interactions that are required to bind the germinants and/or activate the germination machinery.