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.     

Chenodeoxycholate Is an Inhibitor of Clostridium difficile Spore Germination

Some cholate derivatives that are normal components of bile can act with glycine to induce the germination of Clostridium difficile spores, but at least one bile component, chenodeoxycholate, does not induce germination. Here we show that chenodeoxycholate inhibits the germination of C. difficile spores in response to cholate and taurocholate.The anaerobic human pathogen Clostridium difficile must be in the spore form to survive for extended periods of time outside the colonic environment (6). Spores are also the form of the organism most likely to be ingested by a host. To cause disease, however, C. difficile spores must germinate in the gastrointestinal tract and reach the anaerobic environment of the colon, where they can grow out as vegetative bacteria (2). The vegetative form produces two toxins that damage the colonic epithelium and lead to C. difficile-associated diseases, such as diarrhea, pseudomembranous colitis, and toxic megacolon (4, 15). Extending the work of Wilson and colleagues (17, 18), we have shown that certain bile salts and glycine act as cogerminants for C. difficile spores (13). Primary bile salts produced by the liver are composed mainly of cholate (CA) and chenodeoxycholate (CDCA) derivatives conjugated with either taurine or glycine (11). Since CA derivatives are found in the relatively aerobic proximal ileum (9), we reasoned that C. difficile might benefit if its germination were inhibited until the spores reached the anaerobic environment of the large intestine.Inhibitors of germination are typically structurally similar to the germinant whose activities they inhibit. For example, l-alanine-mediated germination of Bacillus subtilis spores is inhibited by d-alanine (16) and 6-thioguanosine inhibits inosine-mediated germination in Bacillus anthracis (1, 16). Since CA and CDCA are structurally similar but CA induces the germination of C. difficile spores (13) and CDCA does not, we tested whether CDCA could act as an inhibitor of germination. C. difficile strain CD196 (10) spores were produced and their concentration determined as described previously (13). After the vegetative bacteria were killed by incubation at 60°C for 20 min, spores were incubated in water containing various concentrations and combinations of bile salts for 10 min. Here we took advantage of the finding by Wilson et al. that C. difficile spores germinate very inefficiently on rich medium plates lacking bile salts (18) unless they are preincubated with bile salts (13, 17). After incubation, spores were serially diluted and plated on brain heart infusion agar supplemented with 5 g yeast extract per liter-0.1% l-cysteine (BHIS) (Difco) in the absence of any bile salt (BHIS contains enough glycine to act as a cogerminant). After overnight growth at 37°C, colonies were enumerated. As a positive or negative control, spores were plated on BHIS containing 0.1% taurocholate (TA) [BHIS(TA)] or on BHIS agar alone, respectively. Preincubation of spores with 0.1% TA in water resulted in the recovery of approximately 0.5% of the total number of spores as colonies compared to results for spores plated directly on BHIS(TA). These results are similar to our previous findings that spores germinate and grow out as colonies more efficiently on agar medium containing TA (13). As reported previously, 0.1% CDCA poorly stimulated colony formation by C. difficile spores (13), yielding only 0.006% spore recovery (Fig. ​(Fig.1A).1A). When TA and CDCA were combined, both at 0.1%, colony formation by C. difficile spores was reduced 21-fold to 0.024% compared to the effect of TA alone. This result indicates that CDCA blocks TA-stimulated colony formation and suggests that CDCA may be an inhibitor of C. difficile spore germination. Increasing the ratio of TA to CDCA suppressed the inhibitory effect of CDCA, increasing colony formation by spores (Fig. ​(Fig.1A).1A). Thus, CDCA seems to block colony formation by competing with TA.Open in a separate windowFIG. 1.CDCA inhibits colony formation by C. difficile spores in response to TA and CA. (A) Spores were prepared and preincubated with TA or CDCA or both in water for 10 min before serial dilution and plating on BHIS agar in the absence of TA. Spores plated on BHIS(TA) served as a positive control for 100% colony formation (CFU). Based on comparisons of total spore counts obtained by microscopy and by colony formation on BHIS(TA) plates, the efficiency of colony formation on BHIS(TA) was estimated at 83%. (B) Spores were prepared as described for panel A and exposed to CA or CDCA or both. Values shown are the averages for three independent experiments, and error bars represent one standard deviation from the mean.CA and other cholate derivatives (e.g., TA, glycocholate, and deoxycholate [DCA]) are also germinants for C. difficile spores (13, 17). To test if CDCA prevents colony formation induced by CA, spores were preexposed to 0.1% CA with and without CDCA. Exposure to CA alone resulted in approximately 1% spore recovery, whereas exposure to 0.1% CA and 0.1% CDCA together led to a decrease in colony formation to 0.075% (Fig. ​(Fig.1B).1B). The effect of CDCA on CA-mediated colony formation was relieved by increasing the concentration of CA to 1.0%, raising colony formation to 2.6% (Fig. ​(Fig.1B).1B). These results indicate that CDCA blocks colony formation induced by CA, as well as that induced by TA, and may be an inhibitor of germination by C. difficile spores that acts competitively in both cases.Spore germination per se is classically measured as a decrease in the optical density of a spore suspension occurring concomitantly with a release of Ca²⁺-dipicolinate from the spore core, rehydration of the core, and degradation of the cortex (8, 12). As determined by this assay, TA is the most effective bile salt for inducing rapid germination (13). To test if CDCA is an inhibitor of germination as opposed to an inhibitor of some other step between germination and colony formation, spores were purified as described previously (13). Spores did not germinate in BHIS medium alone or when this medium was supplemented with 0.1% CDCA (Fig. ​(Fig.2).2). When C. difficile spores were suspended in BHIS containing 0.1% TA, the optical density of the suspension rapidly decreased, indicating that the spores were germinating. However, the optical density of the spores suspended in BHIS with 0.1% TA plus 0.1% CDCA did not decrease over time, indicating that CDCA inhibited TA-mediated germination (Fig. ​(Fig.2).2). When the concentration of TA was increased from 0.1% to 1.0% in the presence of 0.1% CDCA, spores were able to germinate (Fig. ​(Fig.2).2). After overnight incubation in BHIS with 0.1% TA plus 0.1% CDCA, 84% of the spores remained phase bright, while only 11% of spores remained phase bright in BHIS with 1.0% TA plus 0.1% CDCA, indicating that CDCA blocks germination at a very early step. Thus, CDCA is an inhibitor of germination by C. difficile spores that functions by competing with TA and possibly with CA.Open in a separate windowFIG. 2.CDCA inhibits germination of Clostridium difficile spores. Spores were prepared as described previously (13). C. difficile spores were suspended in BHIS alone (•), BHIS plus 0.1% CDCA (▾), BHIS plus 0.1% TA (⧫), BHIS plus 0.1% TA-0.1% CDCA (▪), or BHIS plus 1.0% TA-0.1% CDCA (▴). The ratio of the OD₆₀₀ at the various time points to the OD₆₀₀ at time zero is plotted versus time. Data points are the averages of three independent experiments, and error bars represent one standard deviation from the mean.We previously suggested a role for bile salts in determining the ability of C. difficile to colonize and cause disease (13). In this model, germination of C. difficile spores depends on interaction with glycine and certain bile salts. We show here that the primary bile salt CDCA inhibits germination of C. difficile spores. As mentioned above, germination inhibitors are commonly structurally related to the germinant they inhibit. The structures of CA derivatives and CDCA derivatives are very similar; they differ only insofar as CDCA lacks the 12α hydroxyl group (11).CDCA and CA derivatives are present in approximately equal concentrations in the cecum (5). Under such conditions, CDCA would compete with CA derivatives for binding to putative germinant receptors on C. difficile spores. Mekhjian and colleagues measured the colonic absorption rates of CDCA, CA, and DCA that were introduced into the cecum and collected at the distal colon (7). They found that CDCA was absorbed by the colon at 10 times the rate for CA (7). Thus, when spores reach the distal large intestine, they encounter a decreased ratio of CDCA to CA. Such a change in ratio might allow CA derivatives to act as effective germinants. Thus, C. difficile spores would not be expected to germinate until they reach the colon, which also provides the anaerobic environment required for C. difficile growth.The colonic microflora, which is known to protect the host against C. difficile infection, plays a significant role in the metabolism of bile salts (3, 11). Many different species express on their cell surfaces bile salt hydrolases that serve to remove the conjugated tauryl or glycyl groups from primary bile salts (11). After deconjugation, CA and CDCA are further metabolized by a small percentage of the bacterial species in the cecum to the secondary bile salts deoxycholate and lithocholate, respectively (11, 14). Deoxycholate is an inhibitor of C. difficile growth (13, 17). CDCA inhibits both germination and growth (13). The use of CDCA either as prophylaxis or as a therapy for C. difficile-associated disease might be helpful for patients who are undergoing antibiotic regimens or who are colonized by this bacterium. For example, when an antibiotic that is known to be associated with an increased risk of inciting C. difficile-associated disease is administered, the coadministration of CDCA might protect that individual from colonization by C. difficile through inhibiting spore germination. Alternatively, administering CDCA to individuals who are already being given vancomycin or metronidazole for C. difficile-associated disease may have the benefit of preventing spore germination and further vegetative growth (13) after antibiotic therapy is stopped. This strategy may reduce the already significant risk of a relapse.     

Inhibiting the Initiation of Clostridium difficile Spore Germination using Analogs of Chenodeoxycholic Acid,a Bile Acid

To cause disease, Clostridium difficile spores must germinate in the host gastrointestinal tract. Germination is initiated upon exposure to glycine and certain bile acids, e.g., taurocholate. Chenodeoxycholate, another bile acid, inhibits taurocholate-mediated germination. By applying Michaelis-Menten kinetic analysis to C. difficile spore germination, we found that chenodeoxycholate is a competitive inhibitor of taurocholate-mediated germination and appears to interact with the spores with greater apparent affinity than does taurocholate. We also report that several analogs of chenodeoxycholate are even more effective inhibitors. Some of these compounds resist 7α-dehydroxylation by Clostridium scindens, a core member of the normal human colonic microbiota, suggesting that they are more stable than chenodeoxycholate in the colonic environment.Clostridium difficile is a Gram-positive, spore-forming, anaerobic bacterium that is pathogenic for both humans and animals (33, 44). Infections caused by C. difficile range from mild diarrhea to more life-threatening conditions, such as pseudomembranous colitis (33). In the classic case, prior antibiotic treatment that disrupts the normally protective colonic flora makes patients susceptible to C. difficile infection (CDI) (35, 53). Other antibiotics, such as vancomycin and metronidazole, are the most commonly used treatments for CDI (54). However, because these antibiotics also disrupt the colonic flora, 10 to 40% of patients whose symptoms have been ameliorated suffer from relapsing CDI (15, 24). The annual treatment-associated cost for CDI in the United States is estimated to be between $750 million and $3.2 billion (8, 9, 16, 31). Moreover, the number of fatal cases of CDI has been increasing rapidly (14, 39). Thus, there is an urgent need to find alternative therapies for CDI.C. difficile infection likely is initiated by infection with the spore form of C. difficile (12). C. difficile elicits disease through the actions of two secreted toxins, TcdA and TcdB (48). TcdB was recently shown to be critical for pathogenesis in an animal model of disease (18). Since the toxins are produced by vegetative cells, not by spores (17), germination and outgrowth are prerequisites for pathogenesis.Spore germination is triggered by the interaction of small molecules, called germinants, with receptors within the spore inner membrane. These germinants vary by bacterial species and can include ions, amino acids, sugars, nucleotides, surfactants, or combinations thereof (43). The recognition of germinants triggers irreversible germination, leading to Ca²⁺-dipicolinic acid release, the uptake of water, the degradation of the cortex, and, eventually, the outgrowth of the vegetative bacterium (43). The germination receptors that C. difficile uses to sense the environment have not been identified. Based on homology searches, C. difficile germination receptors must be very different from known germination receptors (42), but they appear to be proteinaceous (13).Taurocholate, a primary bile acid, has been used for approximately 30 years by researchers and clinical microbiologists to increase colony formation by C. difficile spores from patient and environmental samples (3, 49, 51, 52). This suggested that C. difficile spores interact with bile acids along the gastrointestinal (GI) tract and that spores use a host-derived signal to initiate germination.The liver synthesizes the two major primary bile acids, cholate and chenodeoxycholate (40). These compounds are modified by conjugation with either taurine (to give taurocholate or taurochenodeoxycholate) or glycine (producing glycocholate or glycochenodeoxycholate). Upon secretion into the digestive tract, bile aids in the absorption of fat and cholesterol; much of the secreted bile is actively absorbed and recycled back to the liver for reutilization (40). Though efficient, enterohepatic recirculation is not complete; bile enters the cecum of the large intestine at a concentration of approximately 2 mM (30).In the cecum, bile is modified by the normal, benign colonic flora. First, bile salt hydrolases found on the surfaces of many bacterial species remove the conjugated amino acid, producing the deconjugated primary bile acids cholate and chenodeoxycholate (40). These deconjugated primary bile acids are further metabolized by only a few species of intestinal bacteria, including Clostridium scindens. C. scindens actively transports unconjugated primary bile acids into the cytoplasm, where they are 7α-dehydroxylated, converting cholate to deoxycholate and chenodeoxycholate to lithocholate (21, 40). The disruption of the colonic flora by antibiotic treatment abolishes 7α-dehydroxylation activity (41).Building upon the work on Wilson and others (51, 52), we demonstrated that taurocholate and glycine, acting together, trigger the loss of the birefringence of C. difficile spores (45). All cholate derivatives (taurocholate, glycocholate, cholate, and deoxycholate) stimulate the germination of C. difficile spores (45). Recently it was shown that taurocholate binding is prerequisite to glycine binding (37). At physiologically relevant concentrations, chenodeoxycholate inhibits taurocholate-mediated germination (46) and also inhibits C. difficile vegetative growth, as does deoxycholate (45). In fact, C. difficile spores use the relative concentrations of the various bile acids as cues for germination within the host (10).Since chenodeoxycholate is absorbed by the colonic epithelium and metabolized to lithocholate by the colonic flora (25, 40), the use of chenodeoxycholate as a therapy against C. difficile disease might be hindered by its absorption and conversion to lithocholate.Here, we further characterize the interaction of C. difficile spores with various bile acids and demonstrate that chenodeoxycholate is a competitive inhibitor of taurocholate-mediated germination. Further, we identify chemical analogs of chenodeoxycholate that are more potent inhibitors of germination and that resist 7α-dehydroxylation by the colonic flora, potentially increasing their stability and effectiveness as inhibitors of C. difficile spore germination in the colonic environment.     

Activate to Eradicate: Inhibition of Clostridium difficile Spore Outgrowth by the Synergistic Effects of Osmotic Activation and Nisin

Background

Germination is the irreversible loss of spore-specific properties prior to outgrowth. Because germinating spores become more susceptible to killing by stressors, induction of germination has been proposed as a spore control strategy. However, this strategy is limited by superdormant spores that remain unaffected by germinants. Harsh chemicals and heat activation are effective for stimulating germination of superdormant spores but are impractical for use in a hospital setting, where Clostridium difficile spores present a challenge. Here, we tested whether osmotic activation solutes will provide a mild alternative for stimulation of superdormant C. difficile spores in the presence of germinants as previously demonstrated in several species of Bacillus. In addition, we tested the hypothesis that the limitations of superdormancy can be circumvented with a combined approach using nisin, a FDA-approved safe bacteriocin, to inhibit outgrowth of germinated spores and osmotic activation solutes to enhance outgrowth inhibition by stimulating superdormant spores.

Principal Findings

Exposure to germination solution triggered ∼1 log₁₀ colony forming units (CFU) of spores to germinate, and heat activation increased the spores that germinated to >2.5 log₁₀CFU. Germinating spores, in contrast to dormant spores, became susceptible to inhibition by nisin. The presence of osmotic activation solutes did not stimulate germination of superdormant C. difficile spores exposed to germination solution. But, in the absence of germination solution, osmotic activation solutes enhanced nisin inhibition of superdormant spores to >3.5 log₁₀CFU. The synergistic effects of osmotic activation solutes and nisin were associated with loss of membrane integrity.

Conclusions

These findings suggest that the synergistic effects of osmotic activation and nisin bypass the limitations of germination as a spore control strategy, and might be a novel method to safely and effectively reduce the burden of C.difficile spores on skin and environmental surfaces.     

Identification of a Novel Lipoprotein Regulator of Clostridium difficile Spore Germination

Clostridium difficile is a Gram-positive spore-forming pathogen and a leading cause of nosocomial diarrhea. C. difficile infections are transmitted when ingested spores germinate in the gastrointestinal tract and transform into vegetative cells. Germination begins when the germinant receptor CspC detects bile salts in the gut. CspC is a subtilisin-like serine pseudoprotease that activates the related CspB serine protease through an unknown mechanism. Activated CspB cleaves the pro-SleC zymogen, which allows the activated SleC cortex hydrolase to degrade the protective cortex layer. While these regulators are essential for C. difficile spores to outgrow and form toxin-secreting vegetative cells, the mechanisms controlling their function have only been partially characterized. In this study, we identify the lipoprotein GerS as a novel regulator of C. difficile spore germination using targeted mutagenesis. A gerS mutant has a severe germination defect and fails to degrade cortex even though it processes SleC at wildtype levels. Using complementation analyses, we demonstrate that GerS secretion, but not lipidation, is necessary for GerS to activate SleC. Importantly, loss of GerS attenuates the virulence of C. difficile in a hamster model of infection. Since GerS appears to be conserved exclusively in related Peptostreptococcaeace family members, our results contribute to a growing body of work indicating that C. difficile has evolved distinct mechanisms for controlling the exit from dormancy relative to B. subtilis and other spore-forming organisms.     

Triggering Germination Represents a Novel Strategy to Enhance Killing of Clostridium difficile Spores

Background

Clostridium difficile is an anaerobic, spore-forming bacterium that is the most common cause of healthcare-associated diarrhea in developed countries. Control of C. difficile is challenging because the spores are resistant to killing by alcohol-based hand hygiene products, antimicrobial soaps, and most disinfectants. Although initiation of germination has been shown to increase susceptibility of spores of other bacterial species to radiation and heat, it was not known if triggering of germination could be a useful strategy to increase susceptibility of C. difficile spores to radiation or other stressors.

Principal Findings

Here, we demonstrated that exposure of dormant C. difficile spores to a germination solution containing amino acids, minerals, and taurocholic acid resulted in initiation of germination in room air. Germination of spores in room air resulted in significantly enhanced killing by ultraviolet-C (UV-C) radiation and heat. On surfaces in hospital rooms, application of germination solution resulted in enhanced eradication of spores by UV-C administered by an automated room decontamination device. Initiation of germination under anaerobic, but not aerobic, conditions resulted in increased susceptibility to killing by ethanol, suggesting that exposure to oxygen might prevent spores from progressing fully to outgrowth. Stimulation of germination also resulted in reduced survival of spores on surfaces in room air, possibly due to increased susceptibility to stressors such as oxygen and desiccation.

Conclusions

Taken together, these data demonstrate that stimulation of germination could represent a novel method to enhance killing of spores by UV-C, and suggest the possible application of this strategy as a means to enhance killing by other agents.     

Defined Nutrient Diets Alter Susceptibility to Clostridium difficile Associated Disease in a Murine Model

BackgroundClostridium difficile is a major identifiable and treatable cause of antibiotic-associated diarrhea. Poor nutritional status contributes to mortality through weakened host defenses against various pathogens. The primary goal of this study was to assess the contribution of a reduced protein diet to the outcomes of C. difficile infection in a murine model.MethodsC57BL/6 mice were fed a traditional house chow or a defined diet with either 20% protein or 2% protein and infected with C. difficile strain VPI10463. Animals were monitored for disease severity, clostridial shedding and fecal toxin levels. Select intestinal microbiota were measured in stool and C. difficile growth and toxin production were quantified ex vivo in intestinal contents from untreated or antibiotic-treated mice fed with the different diets.ResultsC. difficile infected mice fed with defined diets, particularly (and unexpectedly) with protein deficient diet, had increased survival, decreased weight loss, and decreased overall disease severity. C. difficile shedding and toxin in the stool of the traditional diet group was increased compared with either defined diet 1 day post infection. Mice fed with traditional diet had an increased intestinal Firmicutes to Bacteroidetes ratio following antibiotic exposure compared with either a 2% or 20% protein defined nutrient diet. Ex vivo inoculation of cecal contents from antibiotic-treated mice showed decreased toxin production and C. difficile growth in both defined diets compared with a traditional diet.ConclusionsLow protein diets, and defined nutrient diets in general, were found to be protective against CDI in mice. Associated diet-induced alterations in intestinal microbiota may influence colonization resistance and clostridial toxin production in a defined nutrient diet compared to a traditional diet, leading to increased survival. However, mechanisms which led to survival differences between 2% and 20% protein defined nutrient diets need to be further elucidated.     

Changes in Colonic Bile Acid Composition following Fecal Microbiota Transplantation Are Sufficient to Control Clostridium difficile Germination and Growth

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.     

Spores of Clostridium difficile clinical isolates display a diverse germination response to bile salts

Clostridium difficile spores play a pivotal role in the transmission of infectious diarrhoea, but in order to cause disease spores must complete germination and return to vegetative cell growth. While the mechanisms of spore germination are well understood in Bacillus, knowledge of C. difficile germination remains limited. Previous studies have shown that bile salts and amino acids play an important role in regulating the germination response of C. difficile spores. Taurocholate, in combination with glycine, can stimulate germination, whereas chenodeoxycholate has been shown to inhibit spore germination in a C. difficile clinical isolate. Our recent studies of C. difficile sporulation characteristics have since pointed to substantial diversity among different clinical isolates. Consequently, in this study we investigated how the germination characteristics of different C. difficile isolates vary in response to bile salts. By analysing 29 isolates, including 16 belonging to the BI/NAP1/027 type, we show that considerable diversity exists in both the rate and extent of C. difficile germination in response to rich medium containing both taurocholate and glycine. Strikingly, we also show that although a potent inhibitor of germination for some isolates, chenodeoxycholate does not inhibit the germination, or outgrowth, of all C. difficile strains. Finally, we provide evidence that components of rich media may induce the germination of C. difficile spores, even in the absence of taurocholate. Taken together, these data suggest that the mechanisms of C. difficile spore germination in response to bile salts are complex and require further study. Furthermore, we stress the importance of studying multiple isolates in the future when analysing the nutrients or chemicals that either stimulate or inhibit C. difficile spore germination.     

Proteomic and Genomic Characterization of Highly Infectious Clostridium difficile 630 Spores

Clostridium difficile, a major cause of antibiotic-associated diarrhea, produces highly resistant spores that contaminate hospital environments and facilitate efficient disease transmission. We purified C. difficile spores using a novel method and show that they exhibit significant resistance to harsh physical or chemical treatments and are also highly infectious, with <7 environmental spores per cm² reproducibly establishing a persistent infection in exposed mice. Mass spectrometric analysis identified ∼336 spore-associated polypeptides, with a significant proportion linked to translation, sporulation/germination, and protein stabilization/degradation. In addition, proteins from several distinct metabolic pathways associated with energy production were identified. Comparison of the C. difficile spore proteome to those of other clostridial species defined 88 proteins as the clostridial spore “core” and 29 proteins as C. difficile spore specific, including proteins that could contribute to spore-host interactions. Thus, our results provide the first molecular definition of C. difficile spores, opening up new opportunities for the development of diagnostic and therapeutic approaches.Clostridium difficile is a gram-positive, spore-forming, anaerobic bacterium that can asymptomatically colonize the intestinal tracts of humans and other mammals (3, 30, 39). Antibiotic treatment can result in C. difficile overgrowth and can lead to clinical disease, ranging from diarrhea to life-threatening pseudomembranous colitis, particularly in immunocompromised hosts (2, 4, 7). In recent years, C. difficile has emerged as the major cause of nosocomial antibiotic-induced diarrhea, and it is frequently associated with outbreaks (21, 22). A contributing factor is that C. difficile can be highly infectious and difficult to contain, especially when susceptible patients are present in the same hospital setting (13).Person-to-person transmission of C. difficile is associated with the excretion of highly resistant spores in the feces of infected patients, creating an environmental reservoir that can confound many infection control measures (29, 44). Bacterial spores, which are metabolically dormant cells that are formed following asymmetric cell division, normally have thick concentric external layers, the spore coat and cortex, that protect the internal cytoplasm (15, 42). Upon germination, spores lose their protective external layers and resume vegetative growth (24, 27, 36). Bacillus spores and the spores of most Clostridium species germinate in response to amino acids, carbohydrates, or potassium ions (24, 36). In contrast, C. difficile spores show an increased level of germination in response to cholate derivatives found in bile (40, 41). Thus, spores are well adapted for survival and dispersal under a wide range of environmental conditions but will germinate in the presence of specific molecular signals (24, 36).While the spores of a number of Bacillus species, such as Bacillus subtilis and Bacillus anthracis, and those of other Clostridium species, such as Clostridium perfringens (15, 20), have been well characterized, research on C. difficile spores has been relatively limited. A greater understanding of C. difficile spore biology could be exploited to rationalize disinfection regimes, molecular diagnostics, and the development of targeted treatments such as vaccines. Here we describe a novel method to isolate highly purified C. difficile spores that maintain their resistance and infectious characteristics, thus providing a unique opportunity to study C. difficile spores in the absence of vegetative cells. A thorough proteomic and genomic analysis of the spore provides novel insight into the unique composition and predictive biological properties of C. difficile spores that should underpin future research into this high-profile but poorly understood pathogen.     

Nondigestible Oligosaccharides Enhance Bacterial Colonization Resistance against Clostridium difficile In Vitro

Clostridium difficile is the principal etiologic agent of pseudomembranous colitis and is a major cause of nosocomial antibiotic-associated diarrhea. A limited degree of success in controlling C. difficile infection has been achieved by using probiotics; however, prebiotics can also be used to change bacterial community structure and metabolism in the large gut, although the effects of these carbohydrates on suppression of clostridial pathogens have not been well characterized. The aims of this study were to investigate the bifidogenicity of three nondigestible oligosaccharide (NDO) preparations in normal and antibiotic-treated fecal microbiotas in vitro and their abilities to increase barrier resistance against colonization by C. difficile by using cultural and molecular techniques. Fecal cultures from three healthy volunteers were challenged with a toxigenic strain of C. difficile, and molecular probes were used to monitor growth of the pathogen, together with growth of bifidobacterial and bacteroides populations, over a time course. Evidence of colonization resistance was assessed by determining viable bacterial counts, short-chain fatty acid formation, and cytotoxic activity. Chemostat studies were then performed to determine whether there was a direct correlation between bifidobacteria and C. difficile suppression. NDO were shown to stimulate bifidobacterial growth, and there were concomitant reductions in C. difficile populations. However, in the presence of clindamycin, activity against bifidobacteria was augmented in the presence of NDO, resulting in a further loss of colonization resistance. In the absence of clindamycin, NDO enhanced colonization resistance against C. difficile, although this could not be attributed to bifidobacterium-induced inhibitory phenomena.     

Bile acid-independent protection against Clostridioides difficile infection

Clostridioides difficile infections occur upon ecological / metabolic disruptions to the normal colonic microbiota, commonly due to broad-spectrum antibiotic use. Metabolism of bile acids through a 7α-dehydroxylation pathway found in select members of the healthy microbiota is regarded to be the protective mechanism by which C. difficile is excluded. These 7α-dehydroxylated secondary bile acids are highly toxic to C. difficile vegetative growth, and antibiotic treatment abolishes the bacteria that perform this metabolism. However, the data that supports the hypothesis that secondary bile acids protect against C. difficile infection is supported only by in vitro data and correlative studies. Here we show that bacteria that 7α-dehydroxylate primary bile acids protect against C. difficile infection in a bile acid-independent manner. We monoassociated germ-free, wildtype or Cyp8b1^-/- (cholic acid-deficient) mutant mice and infected them with C. difficile spores. We show that 7α-dehydroxylation (i.e., secondary bile acid generation) is dispensable for protection against C. difficile infection and provide evidence that Stickland metabolism by these organisms consumes nutrients essential for C. difficile growth. Our findings indicate secondary bile acid production by the microbiome is a useful biomarker for a C. difficile-resistant environment but the microbiome protects against C. difficile infection in bile acid-independent mechanisms.     

Clostridium difficile Spore-Macrophage Interactions: Spore Survival

Background

Clostridium difficile is the main cause of nosocomial infections including antibiotic associated diarrhea, pseudomembranous colitis and toxic megacolon. During the course of Clostridium difficile infections (CDI), C. difficile undergoes sporulation and releases spores to the colonic environment. The elevated relapse rates of CDI suggest that C. difficile spores has a mechanism(s) to efficiently persist in the host colonic environment.

Methodology/Principal Findings

In this work, we provide evidence that C. difficile spores are well suited to survive the host’s innate immune system. Electron microscopy results show that C. difficile spores are recognized by discrete patchy regions on the surface of macrophage Raw 264.7 cells, and phagocytosis was actin polymerization dependent. Fluorescence microscopy results show that >80% of Raw 264.7 cells had at least one C. difficile spore adhered, and that ∼60% of C. difficile spores were phagocytosed by Raw 264.7 cells. Strikingly, presence of complement decreased Raw 264.7 cells’ ability to phagocytose C. difficile spores. Due to the ability of C. difficile spores to remain dormant inside Raw 264.7 cells, they were able to survive up to 72 h of macrophage infection. Interestingly, transmission electron micrographs showed interactions between the surface proteins of C. difficile spores and the phagosome membrane of Raw 264.7 cells. In addition, infection of Raw 264.7 cells with C. difficile spores for 48 h produced significant Raw 264.7 cell death as demonstrated by trypan blue assay, and nuclei staining by ethidium homodimer-1.

Conclusions/Significance

These results demonstrate that despite efficient recognition and phagocytosis of C. difficile spores by Raw 264.7 cells, spores remain dormant and are able to survive and produce cytotoxic effects on Raw 264.7 cells.     

Histidine acts as a co‐germinant with glycine and taurocholate for Clostridium difficile spores

Aims: It is well established that the bile salt sodium taurocholate acts as a germinant for Clostridium difficile spores and the amino acid glycine acts as a co‐germinant. The aim of this study was to determine whether any other amino acids act as co‐germinants. Methods and Results: Clostridium difficile spore suspensions were exposed to different germinant solutions comprising taurocholate, glycine and an additional amino acid for 1 h before heating shocking (to kill germinating cells) or chilling on ice. Samples were then re‐germinated and cultured to recover remaining viable cells. Only five amino acids out of the 19 common amino acids tested (valine, aspartic acid, arginine, histidine and serine) demonstrated co‐germination activity with taurocholate and glycine. Of these, only histidine produced high levels of germination (97·9–99·9%) consistently in four strains of Cl. difficile spores. Some variation in the level of germination produced was observed between different PCR ribotypes, and the optimum concentration of amino acids with taurocholate for the germination of Cl. difficile NCTC 11204 spores was 10–100 mmol l^?1. Conclusions: Histidine was found to be a co‐germinant for Cl. difficile spores when combined with glycine and taurocholate. Significance and Impact of the Study: The findings of this study enhance current knowledge regarding agents required for germination of Cl. difficile spores which may be utilized in the development of novel applications to prevent the spread of Cl. difficile infection.     

Bile salts and glycine as cogerminants for Clostridium difficile spores

Spore formation by Clostridium difficile is a significant obstacle to overcoming hospital-acquired C. difficile-associated disease. Spores are resistant to heat, radiation, chemicals, and antibiotics, making a contaminated environment difficult to clean. To cause disease, however, spores must germinate and grow out as vegetative cells. The germination of C. difficile spores has not been examined in detail. In an effort to understand the germination of C. difficile spores, we characterized the response of C. difficile spores to bile. We found that cholate derivatives and the amino acid glycine act as cogerminants. Deoxycholate, a metabolite of cholate produced by the normal intestinal flora, also induced germination of C. difficile spores but prevented the growth of vegetative C. difficile. A model of resistance to C. difficile colonization mediated by the normal bacterial flora is proposed.     

Spore Formation and Toxin Production in Clostridium difficile Biofilms

The ability to grow as a biofilm can facilitate survival of bacteria in the environment and promote infection. To better characterize biofilm formation in the pathogen Clostridium difficile, we established a colony biofilm culture method for this organism on a polycarbonate filter, and analyzed the matrix and the cells in biofilms from a variety of clinical isolates over several days of biofilm culture. We found that biofilms readily formed in all strains analyzed, and that spores were abundant within about 6 days. We also found that extracellular DNA (eDNA), polysaccharide and protein was readily detected in the matrix of all strains, including the major toxins A and/or B, in toxigenic strains. All the strains we analyzed formed spores. Apart from strains 630 and VPI10463, which sporulated in the biofilm at relatively low frequencies, the frequencies of biofilm sporulation varied between 46 and 65%, suggesting that variations in sporulation levels among strains is unlikely to be a major factor in variation in the severity of disease. Spores in biofilms also had reduced germination efficiency compared to spores obtained by a conventional sporulation protocol. Transmission electron microscopy revealed that in 3 day-old biofilms, the outermost structure of the spore is a lightly staining coat. However, after 6 days, material that resembles cell debris in the matrix surrounds the spore, and darkly staining granules are closely associated with the spores surface. In 14 day-old biofilms, relatively few spores are surrounded by the apparent cell debris, and the surface-associated granules are present at higher density at the coat surface. Finally, we showed that biofilm cells possess 100-fold greater resistance to the antibiotic metronidazole then do cells cultured in liquid media. Taken together, our data suggest that C. difficile cells and spores in biofilms have specialized properties that may facilitate infection.     

Germination of Heat- and Alkali-Altered Spores of Clostridium perfringens Type A by Lysozyme and an Initiation Protein

The normal system functioning in the utilization of metabolizable germinants by both heat-sensitive and heat-resistant spores of Clostridium perfringens was inactivated by heat or by treatment of the spores with alkali to remove a soluble coat protein layer. Altered spores were incapable of germination (less than 1%) and outgrowth (less than 0.0005%) in complex media without the addition of either lysozyme or an initiation protein produced by C. perfringens. The addition of either of these agents permitted, in the case of alkali-treated spores, both 90 to 95% germination and outgrowth, as measured by colony formation. In the case of heat-damaged spores, only 50% germination and 2% outgrowth resulted from addition of the initiation protein, whereas lysozyme permitted 85% germination and 8% outgrowth. Alteration of the spores by heat or alkali apparently inactivated the normal lytic system responsible for cortical degradation during germination. Kinetics of production of the initiation protein and conditions affecting both its activity and that of lysozyme on altered spores are described.     

SleC Is Essential for Germination of Clostridium difficile Spores in Nutrient-Rich Medium Supplemented with the Bile Salt Taurocholate

Clostridium difficile is the major cause of infectious diarrhea and a major burden to health care services. The ability of this organism to form endospores plays a pivotal role in infection and disease transmission. Spores are highly resistant to many forms of disinfection and thus are able to persist on hospital surfaces and disseminate infection. In order to cause disease, the spores must germinate and the organism must grow vegetatively. Spore germination in Bacillus is well understood, and genes important for this process have recently been identified in Clostridium perfringens; however, little is known about C. difficile. Apparent homologues of the spore cortex lytic enzyme genes cwlJ and sleB (Bacillus subtilis) and sleC (C. perfringens) are present in the C. difficile genome, and we describe inactivation of these homologues in C. difficile 630Δerm and a B1/NAP1/027 clinical isolate. Spores of a sleC mutant were unable to form colonies when germination was induced with taurocholate, although decoated sleC spores formed the same number of heat-resistant colonies as the parental control, even in the absence of germinants. This suggests that sleC is absolutely required for conversion of spores to vegetative cells, in contrast to CD3563 (a cwlJ/sleB homologue), inactivation of which had no effect on germination and outgrowth of C. difficile spores under the same conditions. The B1/NAP1/027 strain {"type":"entrez-nucleotide","attrs":{"text":"R20291","term_id":"774925","term_text":"R20291"}}R20291 was found to sporulate more slowly and produce fewer spores than 630Δerm. Furthermore, fewer {"type":"entrez-nucleotide","attrs":{"text":"R20291","term_id":"774925","term_text":"R20291"}}R20291 spores germinated, indicating that there are differences in both sporulation and germination between these epidemic and nonepidemic C. difficile isolates.The Gram-positive anaerobe Clostridium difficile causes diarrheal diseases ranging from asymptomatic carriage to a fulminant, relapsing, and potentially fatal colitis (8, 30). This organism is resistant to various broad-spectrum antibiotics and capitalizes on disruption of the normal intestinal flora to colonize and cause disease symptoms through the action of toxins A and B (16, 40). While these toxins are the principal virulence factors, the ability of the organism to produce endospores is necessary for disease transmission.Clostridial spores are extremely resistant to all kinds of chemical and physical agents and provide the mechanism by which C. difficile can evade the potentially fatal consequences of exposure to heat, oxygen, alcohol, and certain disinfectants (35). Thus, the spores shed in fecal matter are very difficult to eradicate and can persist on contaminated surfaces in health care facilities for extended periods of time (35). This leads to infection or reinfection of cohabitating individuals through inadvertent ingestion of infected material (10, 32). Once in the anaerobic environment of the gut, spores presumably germinate to form toxin-producing vegetative cells and, in susceptible individuals, diarrheal disease.Spore germination is defined as the events that result in the irreversible loss of spore characteristics. However, current mechanistic knowledge of the germination process is based principally on data derived from studying Bacillus subtilis. Little is known about spore germination in clostridia and, in particular, in C. difficile. Germination is initiated when the bacterial spore senses specific effectors, termed germinants. These effectors can include nutrients, cationic surfactants, peptidoglycan, and a 1:1 chelate of pyridine-2,6-dicarboxylic acid (dipicolinic acid) and Ca²⁺ (CaDPA) (23, 34, 36). Spores of B. subtilis can germinate in response to nutrients through the participation of three sensory receptors located in the spore inner membrane, GerA, GerB, and GerK (23). After activation, the events include the release of monovalent cations (H⁺, K⁺, and Na⁺) and CaDPA (accounting for approximately 10% of the spore dry weight) (36). The third major step in germination involves hydrolysis of the spore peptidoglycan (PG) cortex. It is during this hydrolysis that the previously low water content of the spore is restored to the water content of a normal vegetative cell and the core is able to expand, which in turn allows enzyme activity, metabolism, and spore outgrowth (36).CwlJ and SleB are two specific spore cortex-lytic enzymes (SCLEs) involved in Bacillus cortex hydrolysis, which break down PG containing muramic-δ-lactam (28). SleB has been shown to localize in both the inner and outer layers of B. subtilis spores through interaction of the enzyme peptidoglycan-binding motif and the δ-lactam structure of the cortex (7, 19) and in association with YpeB, which is required for sleB expression during sporulation (4, 7). SleB is a lytic transglycosylase muramidase, but so far its mode of activation is unknown (21). CwlJ is localized to the spore coat during sporulation (3) and is required for CaDPA-induced germination in B. subtilis. Activation can be due to either CaDPA released from the spore core at the onset of germination or exogenous CaDPA (22). Neither enzyme is individually essential for complete cortex hydrolysis during nutrient germination, although inactivation of both cwlJ and sleB in B. subtilis results in a spore unable to complete this process (15). The role of SleL has recently been studied in Bacillus anthracis. Mutants unable to produce this enzyme are still able to germinate, but the process is retarded (18).The SCLEs of Clostridium are less well studied than those of Bacillus. The SCLEs SleC (20) and SleM (6) have been identified in Clostridium perfringens, and a recent study demonstrated that SleC is required during germination for complete cortex hydrolysis (26). Although SleM can degrade spore cortex peptidoglycan and inactivation of both sleC and sleM decreased the ability of spores to germinate more than inactivation of sleC alone did, SleM was not essential (26). It has also been shown that the germination-specific serine protease CspB is essential for cortex hydrolysis and converts the inactive pro-SleC found in dormant spores to an active enzyme (24). So far, there has been no detailed study of any gene responsible for spore germination in C. difficile, although genes showing homology to cwlJ and sleB of B. subtilis (CD3563) and sleC of C. perfringens (CD0551) have now been identified in the C. difficile 630 genome (33).With germinant receptors in C. difficile yet to be identified, the mechanism by which the spores sense a suitable environment for germination is unclear. Recent studies have suggested that this process may involve the interaction of C. difficile with bile. Taurocholate has been shown to enhance recovery of C. difficile spores in nutrient-rich medium (42), and it has been proposed that glycine and taurocholate act as cogerminants (38), while chenodeoxycholate inhibits C. difficile spore germination (39).The emergence of C. difficile B1/NAP1/027 strains has increased the burden on health care services worldwide. Such strains have been shown to produce higher levels of toxin in the laboratory than many other types of strains (41), although the mechanism behind this production is not fully understood. However, while the observed higher levels of toxin production is doubtless important, perhaps the recent attention given to B1/NAP1/027 strains has focused too much on toxins. As spores represent the infectious stage of C. difficile, processes such as spore germination may also contribute to the greater virulence of these strains. In this study we evaluated the sporulation and germination efficiencies of an “epidemic” B1/NAP1/027 C. difficile strain ({"type":"entrez-nucleotide","attrs":{"text":"R20291","term_id":"774925","term_text":"R20291"}}R20291, isolated from the Stoke Mandeville outbreak in 2004 and 2005) and the “nonepidemic” strain 630Δerm (14). We then constructed strains with mutations in CD3563 (a cwlJ/sleB homologue) and a sleC homologue to analyze the role of these genes in the germination of C. difficile spores.     

An in vitro culture model to study the dynamics of colonic microbiota in Syrian golden hamsters and their susceptibility to infection with Clostridium difficile

Clostridium difficile infections (CDI) are caused by colonization and growth of toxigenic strains of C. difficile in individuals whose intestinal microbiota has been perturbed, in most cases following antimicrobial therapy. Determination of the protective commensal gut community members could inform the development of treatments for CDI. Here, we utilized the lethal enterocolitis model in Syrian golden hamsters to analyze the microbiota disruption and recovery along a 20-day period following a single dose of clindamycin on day 0, inducing in vivo susceptibility to C. difficile infection. To determine susceptibility in vitro, spores of strain VPI 10463 were cultured with and without soluble hamster fecal filtrates and growth was quantified by quantitative PCR and toxin immunoassay. Fecal microbial population changes over time were tracked by 16S ribosomal RNA gene analysis via V4 sequencing and the PhyloChip assay. C. difficile culture growth and toxin production were inhibited by the presence of fecal extracts from untreated hamsters but not extracts collected 5 days post-administration of clindamycin. In vitro inhibition was re-established by day 15, which correlated with resistance of animals to lethal challenge. A substantial fecal microbiota shift in hamsters treated with antibiotics was observed, marked by significant changes across multiple phyla including Bacteroidetes and Proteobacteria. An incomplete return towards the baseline microbiome occurred by day 15 correlating with the inhibition of C. difficile growth in vitro and in vivo. These data suggest that soluble factors produced by the gut microbiota may be responsible for the suppression of C. difficile growth and toxin production.