The Spore Differentiation Pathway in the Enteric Pathogen Clostridium difficile

Endosporulation is an ancient bacterial developmental program that culminates with the differentiation of a highly resistant endospore. In the model organism Bacillus subtilis, gene expression in the forespore and in the mother cell, the two cells that participate in endospore development, is governed by cell type-specific RNA polymerase sigma subunits. σ^F in the forespore, and σ^E in the mother cell control early stages of development and are replaced, at later stages, by σ^G and σ^K, respectively. Starting with σ^F, the activation of the sigma factors is sequential, requires the preceding factor, and involves cell-cell signaling pathways that operate at key morphological stages. Here, we have studied the function and regulation of the sporulation sigma factors in the intestinal pathogen Clostridium difficile, an obligate anaerobe in which the endospores are central to the infectious cycle. The morphological characterization of mutants for the sporulation sigma factors, in parallel with use of a fluorescence reporter for single cell analysis of gene expression, unraveled important deviations from the B. subtilis paradigm. While the main periods of activity of the sigma factors are conserved, we show that the activity of σ^E is partially independent of σ^F, that σ^G activity is not dependent on σ^E, and that the activity of σ^K does not require σ^G. We also show that σ^K is not strictly required for heat resistant spore formation. In all, our results indicate reduced temporal segregation between the activities of the early and late sigma factors, and reduced requirement for the σ^F-to-σ^E, σ^E-to-σ^G, and σ^G-to-σ^K cell-cell signaling pathways. Nevertheless, our results support the view that the top level of the endosporulation network is conserved in evolution, with the sigma factors acting as the key regulators of the pathway, established some 2.5 billion years ago upon its emergence at the base of the Firmicutes Phylum.     

Outpatient Healthcare Settings and Transmission of Clostridium difficile

Background

Recent reports suggest that community-associated Clostridium difficile infection (CDI) (i.e., no healthcare facility admission within 90 days) may be increasing in frequency. We hypothesized that outpatient clinics could be an important source for acquisition of community-associated CDI.

Methods

We performed a 6-month prospective study of CDI patients to determine frequency of and risk factors for skin and environmental shedding during outpatient visits and to derive a prediction rule for positive cultures. We performed a point–prevalence culture survey to assess the frequency of C. difficile contamination in outpatient settings and evaluated the frequency of prior outpatient visits in patients with community-associated CDI.

Results

Of 67 CDI patients studied, 54 (81%) had 1 or more outpatient visits within 12 weeks after diagnosis. Of 44 patients cultured during outpatient visits, 14 (32%) had skin contamination and 12 (27%) contaminated environmental surfaces. Decreased mobility, fecal incontinence, and treatment with non-CDI antibiotics were associated with positive cultures, whereas vancomycin taper therapy was protective. In patients not on CDI therapy, a prediction rule including incontinence or decreased mobility was 90% sensitive and 79% specific for detection of spore shedding. Of 84 clinic and emergency department rooms cultured, 12 (14%) had 1 or more contaminated environmental sites. For 33 community-associated CDI cases, 31 (94%) had an outpatient visit during the 12 weeks prior to onset of diarrhea.

Conclusions

Patients with recent CDI present a significant risk for transmission of spores during outpatient visits. The outpatient setting may be an underappreciated source of community-associated CDI cases.     

Development of Photodynamic Antimicrobial Chemotherapy (PACT) for Clostridium difficile

Background

Clostridium difficile is the leading cause of antibiotic-associated diarrhoea and pseudo membranous colitis in the developed world. The aim of this study was to explore whether Photodynamic Antimicrobial Chemotherapy (PACT) could be used as a novel approach to treating C. difficile infections.

Methods

PACT utilises the ability of light-activated photosensitisers (PS) to produce reactive oxygen species (ROS) such as free radical species and singlet oxygen, which are lethal to cells. We screened thirteen PS against C. difficile planktonic cells, biofilm and germinating spores in vitro, and cytotoxicity of effective compounds was tested on the colorectal adenocarcinoma cell-line HT-29.

Results

Three PS were able to kill 99.9% of bacteria in both aerobic and anaerobic conditions, both in the planktonic state and in a biofilm, after exposure to red laser light (0.2 J/cm²) without harming model colon cells. The applicability of PACT to eradicate C. difficile germinative spores indirectly was also shown, by first inducing germination with the bile salt taurocholate, followed by PACT.

Conclusion

This innovative and simple approach offers the prospect of a new antimicrobial therapy using light to treat C. difficile infection of the colon.     

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.     

Variations in TcdB Activity and the Hypervirulence of Emerging Strains of Clostridium difficile

Hypervirulent strains of Clostridium difficile have emerged over the past decade, increasing the morbidity and mortality of patients infected by this opportunistic pathogen. Recent work suggested the major C. difficile virulence factor, TcdB, from hypervirulent strains (TcdB_HV) was more cytotoxic in vitro than TcdB from historical strains (TcdB_HIST). The current study investigated the in vivo impact of altered TcdB tropism, and the underlying mechanism responsible for the differences in activity between the two forms of this toxin. A combination of protein sequence analyses, in vivo studies using a Danio rerio model system, and cell entry combined with fluorescence assays were used to define the critical differences between TcdB_HV and TcdB_HIST. Sequence analysis found that TcdB was the most variable protein expressed from the pathogenicity locus of C. difficile. In line with these sequence differences, the in vivo effects of TcdB_HV were found to be substantially broader and more pronounced than those caused by TcdB_HIST. The increased toxicity of TcdB_HV was related to the toxin''s ability to enter cells more rapidly and at an earlier stage in endocytosis than TcdB_HIST. The underlying biochemical mechanism for more rapid cell entry was identified in experiments demonstrating that TcdB_HV undergoes acid-induced conformational changes at a pH much higher than that of TcdB_HIST. Such pH-related conformational changes are known to be the inciting step in membrane insertion and translocation for TcdB. These data provide insight into a critical change in TcdB activity that contributes to the emerging hypervirulence of C. difficile.     

Evaluation of an Automated Rapid Diagnostic Test for Detection of Clostridium difficile

The Verigene Clostridium difficile Nucleic Acid Test (Verigene CDF Test) (Nanosphere, Northbrook, IL, USA) is a new multiplex qualitative polymerase chain reaction (PCR) test used to detect C. difficile toxin genes in fecal specimens. To evaluate the performance of the new method, we tested 69 fecal samples from patients with suspected C. difficile infection using the Verigene CDF test, an enzyme immunoassay (EIA) and PCR following anaerobic fecal culture. The sensitivity, specificity, and accuracy of the Verigene CDF test were 96.7% (29/30), 97.4% (38/39), and 97.1% (67/69) respectively, using PCR following fecal culture as a reference method. We also analyzed the potential clinical impact of the Verigene CDF test using chart reviews of the 69 patients with suspected C. difficile infection and found that 11 of the 69 patients were incorrectly diagnosed, and the Verigene CDF test would have led to them receiving more appropriate management including practice of treatment and contact precaution, although, of the 69 patients, there are two whose samples were incorrectly identified with the Verigene CDF test. The Verigene CDF test will have a positive impact on patient care.     

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.     

Succession in the Gut Microbiome following Antibiotic and Antibody Therapies for Clostridium difficile

Antibiotic disruption of the intestinal microbiota may cause susceptibility to pathogens that is resolved by progressive bacterial outgrowth and colonization. Succession is central to ecological theory but not widely documented in studies of the vertebrate microbiome. Here, we study succession in the hamster gut after treatment with antibiotics and exposure to Clostridium difficile. C. difficile infection is typically lethal in hamsters, but protection can be conferred with neutralizing antibodies against the A and B toxins. We compare treatment with neutralizing monoclonal antibodies (mAb) to treatment with vancomycin, which prolongs the lives of animals but ultimately fails to protect them from death. We carried out longitudinal deep sequencing analysis and found distinctive waves of succession associated with each form of treatment. Clindamycin sensitization prior to infection was associated with the temporary suppression of the previously dominant Bacteroidales and the fungus Saccinobaculus in favor of Proteobacteria. In mAb-treated animals, C. difficile proliferated before joining Proteobacteria in giving way to re-expanding Bacteroidales and the fungus Wickerhamomyces. However, the Bacteroidales lineages returning by day 7 were different from those that were present initially, and they persisted for the duration of the experiment. Animals treated with vancomycin showed a different set of late-stage lineages that were dominated by Proteobacteria as well as increased disparity between the tissue-associated and luminal cecal communities. The control animals showed no change in their gut microbiota. These data thus suggest different patterns of ecological succession following antibiotic treatment and C. difficile infection.     

Spo0A Differentially Regulates Toxin Production in Evolutionarily Diverse Strains of Clostridium difficile

Clostridium difficile is an important pathogen of humans and animals, representing a significant global healthcare problem. The last decade has seen the emergence of epidemic BI/NAP1/027 and ribotype 078 isolates, associated with the onset of more severe disease and higher rates of morbidity and mortality. However, little is known about these isolates at the molecular level, partly due to difficulties in the genetic manipulation of these strains. Here we report the development of an optimised Tn916-mediated plasmid transfer system, and the use of this system to construct and complement spo0A mutants in a number of different C. difficile strain backgrounds. Spo0A is a global regulator known to control sporulation, but may also be involved in the regulation of potential virulence factors and other phenotypes. Recent studies have failed to elucidate the role of Spo0A in toxin A and toxin B production by C. difficile, with conflicting data published to date. In this study, we aimed to clarify the role of Spo0A in production of the major toxins by C. difficile. Through the construction and complementation of spo0A mutants in two ribotype 027 isolates, we demonstrate that Spo0A acts as a negative regulator of toxin A and toxin B production in this strain background. In addition, spo0A was disrupted and subsequently complemented in strain 630Δerm and, for the first time, in a ribotype 078 isolate, JGS6133. In contrast to the ribotype 027 strains, Spo0A does not appear to regulate toxin production in strain 630Δerm. In strain JGS6133, Spo0A appears to negatively regulate toxin production during early stationary phase, but has little effect on toxin expression during late stationary phase. These data suggest that Spo0A may differentially regulate toxin production in phylogenetically distinct C. difficile strain types. In addition, Spo0A may be involved in regulating some aspects of C. difficile motility.     

Surveillance of Infection Severity: A Registry Study of Laboratory Diagnosed Clostridium difficile

Background

Changing clinical impact, as virulent clones replace less virulent ones, is a feature of many pathogenic bacterial species and can be difficult to detect. Consequently, innovative techniques monitoring infection severity are of potential clinical value.

Methods and Findings

We studied 5,551 toxin-positive and 20,098 persistently toxin-negative patients tested for Clostridium difficile infection between February 1998 and July 2009 in a group of hospitals based in Oxford, UK, and investigated 28-day mortality and biomarkers of inflammation (blood neutrophil count, urea, and creatinine concentrations) collected at diagnosis using iterative sequential regression (ISR), a novel joinpoint-based regression technique suitable for serial monitoring of continuous or dichotomous outcomes. Among C. difficile toxin-positive patients in the Oxford hospitals, mean neutrophil counts on diagnosis increased from 2003, peaked in 2006–2007, and then declined; 28-day mortality increased from early 2006, peaked in late 2006–2007, and then declined. Molecular typing confirmed these changes were likely due to the ingress of the globally distributed severe C. difficile strain, ST1. We assessed the generalizability of ISR-based severity monitoring in three ways. First, we assessed and found strong (p<0.0001) associations between isolation of the ST1 severe strain and higher neutrophil counts at diagnosis in two unrelated large multi-centre studies, suggesting the technique described might be useful elsewhere. Second, we assessed and found similar trends in a second group of hospitals in Birmingham, UK, from which 5,399 cases were analysed. Third, we used simulation to assess the performance of this surveillance system given the ingress of future severe strains under a variety of assumptions. ISR-based severity monitoring allowed the detection of the severity change years earlier than mortality monitoring.

Conclusions

Automated electronic systems providing early warning of the changing severity of infectious conditions can be established using routinely collected laboratory hospital data. In the settings studied here these systems have higher performance than those monitoring mortality, at least in C. difficile infection. Such systems could have wider applicability for monitoring infections presenting in hospital.     

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.     

Deoxyribonucleic Acid Synthesis and Deoxynucleotide Metabolism During Bacterial Spore Germination

Deoxyribonucleic acid (DNA) synthesis during germination of Bacillus megaterium spores takes place in two stages. In stage I (0-55 min) DNA synthesis is slow and there is no detectable net synthesis, whereas in stage II (from 55 min on) the rate of synthesis is much faster and net DNA synthesis occurs. Deoxyribonucleotide pool sizes match the rates of DNA synthesis in stages I and II. The level of deoxyribonucleotide triphosphates is not correlated with the level of deoxyribonucleotide kinases, but rather with that of ribonucleotide reductase activity.     

Surface-Layer Protein A (SlpA) Is a Major Contributor to Host-Cell Adherence of Clostridium difficile

Clostridium difficile is a leading cause of antibiotic-associated diarrhea, and a significant etiologic agent of healthcare-associated infections. The mechanisms of attachment and host colonization of C. difficile are not well defined. We hypothesize that non-toxin bacterial factors, especially those facilitating the interaction of C. difficile with the host gut, contribute to the initiation of C. difficile infection. In this work, we optimized a completely anaerobic, quantitative, epithelial-cell adherence assay for vegetative C. difficile cells, determined adherence proficiency under multiple conditions, and investigated C. difficile surface protein variation via immunological and DNA sequencing approaches focused on Surface-Layer Protein A (SlpA). In total, thirty-six epidemic-associated and non-epidemic associated C. difficile clinical isolates were tested in this study, and displayed intra- and inter-clade differences in attachment that were unrelated to toxin production. SlpA was a major contributor to bacterial adherence, and individual subunits of the protein (varying in sequence between strains) mediated host-cell attachment to different extents. Pre-treatment of host cells with crude or purified SlpA subunits, or incubation of vegetative bacteria with anti-SlpA antisera significantly reduced C. difficile attachment. SlpA-mediated adherence-interference correlated with the attachment efficiency of the strain from which the protein was derived, with maximal blockage observed when SlpA was derived from highly adherent strains. In addition, SlpA-containing preparations from a non-toxigenic strain effectively blocked adherence of a phylogenetically distant, epidemic-associated strain, and vice-versa. Taken together, these results suggest that SlpA plays a major role in C. difficile infection, and that it may represent an attractive target for interventions aimed at abrogating gut colonization by this pathogen.     

High Molecular Weight Typing with MALDI-TOF MS - A Novel Method for Rapid Typing of Clostridium difficile

Clostridium difficile strains were typed by a newly developed MALDI-TOF method, high molecular weight typing, and compared to PCR ribotyping. Among 500 isolates representing 59 PCR ribotypes a total of 35 high molecular weight types could be resolved. Although less discriminatory than PCR ribotyping, the method is extremely fast and simple, and supports for cost-effective screening of isolates during outbreak situations.     

Functional Characterisation of Germinant Receptors in Clostridium botulinum and Clostridium sporogenes Presents Novel Insights into Spore Germination Systems

Clostridium botulinum is a dangerous pathogen that forms the highly potent botulinum toxin, which when ingested causes a deadly neuroparalytic disease. The closely related Clostridium sporogenes is occasionally pathogenic, frequently associated with food spoilage and regarded as the non-toxigenic equivalent of Group I C. botulinum. Both species form highly resistant spores that are ubiquitous in the environment and which, under favourable growth conditions germinate to produce vegetative cells. To improve the control of botulinum neurotoxin-forming clostridia, it is imperative to comprehend the mechanisms by which spores germinate. Germination is initiated following the recognition of small molecules (germinants) by a specific germinant receptor (GR) located in the spore inner membrane. The present study precisely defines clostridial GRs, germinants and co-germinants. Group I C. botulinum ATCC3502 contains two tricistronic and one pentacistronic GR operons, while C. sporogenes ATCC15579 has three tricistronic and one tetracistronic GR operons. Insertional knockout mutants, allied with characterisation of recombinant GRs shows for the first time that amino acid stimulated germination in C. botulinum requires two tri-cistronic encoded GRs which act in synergy and cannot function individually. Spore germination in C. sporogenes requires one tri-cistronic GR. Two other GRs form part of a complex involved in controlling the rate of amino-acid stimulated germination. The suitability of using C. sporogenes as a substitute for C. botulinum in germination studies and food challenge tests is discussed.     

The SOS Response Master Regulator LexA Is Associated with Sporulation,Motility and Biofilm Formation in Clostridium difficile

The LexA regulated SOS network is a bacterial response to DNA damage of metabolic or environmental origin. In Clostridium difficile, a nosocomial pathogen causing a range of intestinal diseases, the in-silico deduced LexA network included the core SOS genes involved in the DNA repair and genes involved in various other biological functions that vary among different ribotypes. Here we describe the construction and characterization of a lexA ClosTron mutant in C. difficile {"type":"entrez-nucleotide","attrs":{"text":"R20291","term_id":"774925"}}R20291 strain. The mutation of lexA caused inhibition of cell division resulting in a filamentous phenotype. The lexA mutant also showed decreased sporulation, a reduction in swimming motility, greater sensitivity to metronidazole, and increased biofilm formation. Changes in the regulation of toxin A, but not toxin B, were observed in the lexA mutant in the presence of sub-inhibitory concentrations of levofloxacin. C. difficile LexA is, therefore, not only a regulator of DNA damage but also controls many biological functions associated with virulence.