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.     

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.     

SpoIVA and SipL Are Clostridium difficile Spore Morphogenetic Proteins

Clostridium difficile is a major nosocomial pathogen whose infections are difficult to treat because of their frequent recurrence. The spores of C. difficile are responsible for these clinical features, as they resist common disinfectants and antibiotic treatment. Although spores are the major transmissive form of C. difficile, little is known about their composition or morphogenesis. Spore morphogenesis has been well characterized for Bacillus sp., but Bacillus sp. spore coat proteins are poorly conserved in Clostridium sp. Of the known spore morphogenetic proteins in Bacillus subtilis, SpoIVA is one of the mostly highly conserved in the Bacilli and the Clostridia. Using genetic analyses, we demonstrate that SpoIVA is required for proper spore morphogenesis in C. difficile. In particular, a spoIVA mutant exhibits defects in spore coat localization but not cortex formation. Our study also identifies SipL, a previously uncharacterized protein found in proteomic studies of C. difficile spores, as another critical spore morphogenetic protein, since a sipL mutant phenocopies a spoIVA mutant. Biochemical analyses and mutational analyses indicate that SpoIVA and SipL directly interact. This interaction depends on the Walker A ATP binding motif of SpoIVA and the LysM domain of SipL. Collectively, these results provide the first insights into spore morphogenesis in C. difficile.     

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.     

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.     

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.     

Clostridium difficile electrophoretic typing

Abstract Two typing schemes for Clostridium difficile based on slide agglutinations and polyacrylamide gel electrophoresis (PAGE) have been described. We compared the reference strains of each typing system with a simplified PAGE method using whole cells and Coomassie blue staining. The method was also applied to clinical isolates and immunoblots were performed with a monospecific serum directed against a major band of low molecular weight. The results indicated the great heterogeneity of Clostridium difficile strains complicated by antigenic subdivision for strains belonging to the same electrophoretic type.     

Clostridium sordellii bacteriophage active on Clostridium difficile

Among five strains of Clostridium difficile and 39 strains of Cl. sordellii tested, one Cl. difficile phage and four Cl. sordellii phages were found to be lytic for Cl. difficille strain 2. The five phages were similar in morphology, showing a polyhedral head of 60 nm in diameter, a tail of 105–120 nm, a contractile tail sheath and a base plate. They were sensitive to heat (60°C/10 min) and stable at 4°C for at least 6 months. As the phage donor strains and the indicator strain were not cytotoxigenic, no phage-infected culture of Cl. difficile 2 was able to produce cytotoxin.     

Antibiotics and Clostridium difficile

Clostridium difficile is now established as a major nosocomial pathogen. C. difficile infection is seen almost exclusively as a complication of antibiotic therapy, and is particularly associated with clindamycin and third-generation cephalosporins. Depletion of the indigenous gut microflora by antibiotic therapy has long been established as a major factor in the disease. However, the direct influence of antimicrobials upon virulence mechanisms such as toxin production and adhesion in the bowel, and the exact mechanisms by which the organism causes disease remain to be elucidated.     

Spore Fine Structure in Clostridium cochlearium

The fine structure of Clostridium cochlearium was examined by use of thin sections, negative stains, and carbon replicas. Particular attention was given to details of the sporulation process and to fine structure of the spores. Spore coat formation was well advanced before the first evidence of cortex formation was noted. Three distinct spore coats were detected, the outermost of which was composed of seven layers. In addition, the spores possessed tubular appendages of variable length attached to one end of the spore. These differed in a number of respects from those described for other clostridia.     

Thiosemicarbazones active against Clostridium difficile

A set of closely related furylidene thiosemicarbazones was prepared and screened against various clinically important Gram-positive bacteria. One compound containing an ethylene spacer and a 5-nitrofuryl group was found to have promising activity against Clostridium difficile.     

Clostridium difficile in emergency room

Clostridium difficile strains are known as etiological agents of pseudomembranous colitis (PMC), antibiotic-associated diarrhea (AAC) and colitis (AAC) and hospital-acquired infections. The aim of this study was to determine the frequency of C. difficile infection among patients in the emergency room and to compare isolated strains by phenotypic and genotypic characteristics. During a period of 11 months, 56 stool samples taken from diarrheic patients hospitalized in the emergency room of the Medical Center UC Davis and 14 environmental samples were cultured for isolation of C. difficile strains. Eighteen C. difficile strains were isolated from stool samples cultured on selective TCCCA plates and 5 strains from environmental samples using Rodac plates. Eleven toxigenic (TcdA+/TcdB+), 6 non-toxigenic (TcdA-/TcdB-) and unique toxin A-negative/toxin B-positive (TcdA-/TcdB+) C. difficile strains were detected among patients' isolates and 3 toxigenic and 2 non-toxigenic strains-among environmental samples. The majority of C. difficile-positive patients were treated previously by antibiotics. Four strains isolated from patients' fecal samples and one strain isolated from the environment demonstrated high-level resistance to erythromycin and clindamycin (MIC >256mug/mL). The results obtained by AP-PCR and PCR-ribotyping revealed genetic heterogeneity among the strains isolated from patients' fecal samples. However, similarity was observed among environmental strains and strains isolated from patients' fecal samples. Considering the importance of emergency room patients as a potential source of C. difficile strains, it appears to be important examine these patients for C. difficile before transfer to the other hospital units.     

Sporulation studies in Clostridium difficile

Clostridium difficile is a leading cause of healthcare-associated diarrhoea. In recent years, certain C. difficile types have become highly represented among clinical isolates and are associated with outbreaks of increased disease severity, higher relapse rates and an expanded repertoire of antibiotic resistance. Endospores, produced during sporulation, play a pivotal role in infection and disease transmission and it has been suggested in the literature that these so-called ‘hypervirulent’ C. difficile types are more prolific in terms of sporulation in vitro. However, work in our laboratory has provided evidence to the contrary suggesting that although there is significant strain-to-strain variation in C. difficile sporulation characteristics this variation does not appear to be type-associated. On analysis of the literature, it is apparent that the methods used to quantify sporulation in previous studies have varied greatly and sample sizes have remained small. The conflicting data in the literature may, therefore, not necessarily be generally representative of C. difficile sporulation. Instead, these inconsistencies may reflect differences in the experimental design of each study. In this review, the need for further investigations of C. difficile sporulation rates is highlighted. Specifically, the advantages and disadvantages of the different experimental approaches previously used are discussed and a standard set of principles for measuring C. difficile sporulation in the future is proposed.     

Carbohydrate fermentation by Clostridium difficile

Biochemical properties of Clostridium difficile were reinvestigated for the practical identification of the organism in clinical laboratories. Bacterial growth in 2% proteose peptone medium supplemented with 0.01% L-cysteine.HCl and 0.1% agar supported sufficient growth to read the fermentation results just as well as did pre-reduced anaerobically sterilized medium. Incubation for 2 days was long enough for determining the ability to ferment fructose, glucose, mannitol, mannose, melezitose, and sorbitol. All of the 82 strains liquefied 2% but not 10% gelatin. The significance of mannitol fermentation and gelatin liquefaction is stressed since C. difficile is the only species fermenting mannitol among the gelatin-liquefying species of clostridia having subterminal spores.