- Volume 57, Issue 6, 2008

The pathogenicity of Clostridium difficile depends on the large clostridial glucosylating toxins A and B (TcdA and TcdB). The proteins accomplish their own uptake by a modular structure comprising a catalytic and a binding/translocation domain. Based on a proteolytic processing step solely the catalytic domain reaches the cytosol. Within the cells, the glucosyltransferases inactivate small GTPases by mono-O-glucosylation. Here, a short overview is given regarding latest insights into the intramolecular processing, which is mediated by an intrinsic protease activity.

The prevalence of toxigenic Clostridium difficile in Korea has been reported to be approximately 60–80 %. Although the prevalence of the tcdA−tcdB+ C. difficile strain was less then 5 % prior to the year 2000, it has become an emerging nosocomial pathogen in Korea. Therefore, we have attempted to determine the multicentre nationwide prevalence of tcdA+tcdB+ and tcdA−tcdB+ C. difficile for epidemiological purposes. C. difficile strains (n=724, 30 from 2000, 80 from 2001, 74 from 2002, 76 from 2003, 179 from 2004, 285 from 2005) were obtained retrospectively from January 2000 to December 2005 from in-patients at 6 hospitals, all of whom were suspected of having C. difficile-associated disease (CDAD), colitis or pseudomembranous colitis. The numbers of participating hospitals varied yearly (1 in 2000, 2 in 2001–2003, 3 in 2004, 5 in 2005). The hospitals were located in Seoul (n=4), Kyunggi Province (n=1) and Busan (n=1), Korea. PCR assays for tcdA and tcdB genes were conducted using 724 unduplicated C. difficile isolates. The mean prevalence of tcdA+tcdB+ and tcdA−tcdB+ C. difficile strains over the 6 years was 51.8 % (38.4–59.3 %) and 25.8 %(10–56.0 %), respectively. The mean prevalence of tcdA−tcdB+ C. difficile strains was less than 7 % until 2002, but began to increase in 2003 (13.2 %) and achieved a peak in 2004 (50.3 %). In 2005, the mean prevalence of tcdA+tcdB+ and tcdA−tcdB+ C. difficile strains was 47.7 % (30.9–60.3 %) and 27.0 % (17.6–54.8 %), respectively. This nationwide epidemiological study showed that tcdA−tcdB+ C. difficile strains have already spread extensively throughout Korea, and our results provide basic data regarding the controversies currently surrounding the toxigenicity of tcdA−tcdB+ C. difficile. The use of enzyme immunoassays capable of detecting both TcdA and TcdB is strongly recommended for the diagnosis of CDAD in microbiology laboratories, in order to control the spread of the tcdA−tcdB+ strains of C. difficile.

In order to assess the lethality of Clostridium difficile-associated disease (CDAD) and the PCR ribotypes prevalent in Austria, the Austrian Agency for Health and Food Safety requested isolates of C. difficile from patients in a structured but arbitrary sampling scheme. In the allocated period from February 2006 to January 2007, local hospital laboratories within each of the nine provinces were asked to submit C. difficile isolates from at least ten cases of CDAD. Confirmation of species identification, toxin detection, susceptibility testing against four antimicrobial agents and typing using a PCR ribotyping method were performed at the reference laboratory. In total, 149 isolates of putative C. difficile were submitted, from which 142 were included for study. Antimicrobial susceptibility patterns revealed resistance to clindamycin in 57 % and high-level resistance to moxifloxacin in 38 % of isolates tested. CDAD manifested as diarrhoea (including eight cases of bloody diarrhoea) in 126 cases (88.7 %), as pseudomembranous colitis in 15 cases (10.6 %) and as toxic megacolon in one case. Twelve of the 142 patients died within 30 days of specimen collection (8.45 % lethality). A lethal outcome occurred in 2/15 cases (13.3 %) when pseudomembranous colitis was present and in 10/126 cases (7.9 %) in the absence of pseudomembranous colitis or toxic megacolon. Among the 142 isolates from 25 health-care facilities, 41 PCR ribotype patterns were found. The most frequent ribotypes were AI-5 (including six lethal cases out of 26 patients), 014 (two out of 24) and 053 (one out of 24). The typing patterns demonstrated the occurrence of clusters in hospitals.

The first Dutch outbreak due to Clostridium difficile ribotype 027 was observed in mid-2005; by the end of that year, eight hospitals were affected. To study the relationship between hospital-wide antibiotic use and the incidence of 027-linked C. difficile-associated disease (CDAD) three study groups were made: group A, all eight hospitals with an 027-associated epidemic; group B, five of a total of six hospitals with occasional 027 cases, without an increase in CDAD; and group C, ten randomly selected hospitals with no reported 027 epidemics or isolated 027 cases. Quarterly data on CDAD incidences, hygiene measures and the use of fluoroquinolones, second- and third-generation cephalosporins, extended-spectrum penicillins, penicillins with beta-lactamase inhibitors, carbapenems, lincomycins and macrolides were collected for 2004 and 2005, and divided into pre-epidemic and epidemic periods. Using a multilevel Poisson regression analysis, CDAD incidence was linked to antibiotic use in the previous quarter and to certain hygiene measures. In the pre-epidemic period, the total use of the studied antibiotics was comparable between affected and unaffected hospitals. Higher use of second-generation cephalosporins, macrolides and all of the studied antibiotics were independently associated with a small increase in CDAD incidence [relative risk (95 % confidence interval): 1.14 per increase of 100 defined daily doses per 10 000 bed days (1.06–1.23), 1.10 (1.01–1.19) and 1.02 (1.01–1.03), respectively]. However the effect was too small to predict which hospitals might be more prone to 027-associated outbreaks.

It has been proposed that patients who develop Clostridium difficile-associated disease (CDAD) do so because they are unable to mount an adequate immune response. Serum was collected from three groups of elderly in-patients: (i) cases (n=21) of CDAD, being toxin A/B-positive; (ii) carriers (n=21) asymptomatic for CDAD (no diarrhoea) but at least toxin or culture positive; and (iii) controls (n=26) asymptomatic for CDAD and negative for both C. difficile toxin and culture. The age and gender of each group were compared, and the colonizing strains were ribotyped and toxinotyped. Serum antibodies (IgG and IgM) were measured by ELISA using different antigen preparations: EDTA extract (containing cell-surface proteins and carbohydrates), guanidine hydrochloride extract (surface-layer proteins), aqueous phenol-extracted lipocarbohydrate (LC); crude toxin (dialysis culture supernatant) and purified toxin A. LPS from Escherichia coli was used as a control antigen. Antibodies were also tested for toxin neutralization on tissue monolayers and for binding to EDTA-extracted antigens by Western blotting. IgG antibody measurements to cytomegalovirus (CMV) were included as an indicator of potential immunosenescence. Results showed that the patient groups were well matched by age and gender, and the colonizing strains were similar in cases and carriers, being predominantly ribotype 001 and toxinotype 0. By ELISA, IgG levels to most of the antigens were highest in the cases and lowest in the controls, with the exception of antibodies to the LC, which were higher in the controls than the cases. Levels in the carriers tended to be of intermediate level or similar to the controls. For all antigens, the levels of IgM were not significantly different among cases, carriers and controls. Serum from all groups was able to neutralize the cytotoxic action of toxin on both Vero and Caco2 cells, and all to a similar extent. Western blots showed an overall higher level of IgG antibodies to the EDTA-extracted antigens in the cases. The results of the CMV ELISA showed that specific IgG was detected in more cases (78 %) than carriers and controls (both 65 %), but this difference in seropositivity was not significant. The conclusion is that, during symptomatic infection, patients respond to protein antigens of C. difficile in a manner typical of a secondary antibody response, with no evidence that an inability to respond predisposes to the appearance of symptoms.

This study evaluated the in vivo adjuvant activity of two peptides derived from Clostridium difficile: a fragment of the receptor-binding domain of toxin A (TxAC314) and a fragment of the 36 kDa surface-layer protein (SLP-36kDa) from strain C253. Their ability to affect the magnitude, distribution and polarization of the immune response against fibronectin-binding protein A (FnbpA), a protective vaccine antigen against Staphylococcus aureus, was evaluated using two different routes of immunization: intranasal and subcutaneous. It was shown that (i) the route of immunization affected the magnitude of the immune response; (ii) both peptides enhanced the production of circulating anti-FnbpA IgG and IgA; (iii) following mucosal immunization TxAC314 was more effective than SLP-36kDa at inducing antibody in the gastrointestinal tract; (iv) the adjuvant influenced the Th1/Th2 balance; and (v) TxAC314 was more effective than SLP-36kDa in inducing a cell-mediated response. These studies provide insight into the ability of different C. difficile-derived peptides to differentially affect and polarize the activity of the immune system and on their potential use as adjuvants in newly developed vaccines.

Clostridium difficile is the most common cause of antibiotic-associated diarrhoea. Antibiotics are presumed to disturb the normal intestinal microbiota, leading to depletion of the barrier effect and colonization by pathogenic bacteria. This first step of infection includes adherence to epithelial cells. We investigated the impact of various environmental conditions in vitro on the expression of genes encoding known, or putative, colonization factors: three adhesins, P47 (one of the two S-layer proteins), Cwp66 and Fbp68, and a protease, Cwp84. The conditions studied included hyperosmolarity, iron depletion and exposure to several antibiotics (ampicillin, clindamycin, ofloxacin, moxifloxacin and kanamycin). The analysis was performed on three toxigenic and three non-toxigenic C. difficile isolates using real-time PCR. To complete this work, the impact of ampicillin and clindamycin on the adherence of C. difficile to Caco-2/TC7 cells was analysed. Overall, for the six strains of C. difficile studied, exposure to subinhibitory concentrations (1/2 MIC) of clindamycin and ampicillin led to the increased expression of genes encoding colonization factors. This was correlated with the increased adherence of C. difficile to cultured cells under the same conditions. The levels of gene regulation observed among the six strains studied were highly variable, cwp84 being the most upregulated. In contrast, the expression of these genes was weakly, or not significantly, modified in the presence of ofloxacin, moxifloxacin or kanamycin. These results suggest that, in addition to the disruption of the normal intestinal microbiota and its barrier effect, the high propensity of antibiotics such as ampicillin and clindamycin to induce C. difficile infection could also be explained by their direct role in enhancing colonization by C. difficile.

Cell-surface antigens of Clostridium difficile and LPS from Escherichia coli were investigated for modulating effects on the activity of C. difficile toxin A on Vero and Caco2 cells. The antigens of C. difficile tested comprised: (i) an EDTA extract, which contained several major and minor cell-surface proteins and the membrane-associated lipocarbohydrate (LC); (ii) a guanidine hydrochloride extract, which mainly contained the surface-layer proteins; (iii) an aqueous phenol-extracted, protein-free LC. On their own, none of the antigens had a detrimental effect on the cells, with the EDTA extract and LC having a marginally protective effect. When these antigens were added to suboptimal levels of toxin A, there was significant enhancement of its cytotoxicity by the EDTA and LC preparations on both cell types. LPS showed some enhancement of the effect of toxin on Vero cells at the lowest levels of toxin investigated. It was concluded that this effect seen in vitro may have a role to play in the colon during infection with C. difficile.

Clostridium difficile is an important nosocomial pathogen, resulting in antibiotic-associated disease ranging from mild diarrhoea to the life-threatening pseudomembranous colitis. Upon antibiotic exposure, it is believed that the normal bowel microflora of patients is disrupted, allowing C. difficile to proliferate. Significantly, C. difficile is among only a few bacteria able to ferment tyrosine to p-cresol, a phenolic compound that is toxic to other microbes via its ability to interfere with metabolism. Therefore, the ability of different C. difficile strains to produce and tolerate p-cresol may play an important role in the development and severity of C. difficile-associated disease. In this study, it was demonstrated that two C. difficile hypervirulent 027 strains (Stoke Mandeville and BI-16) are more tolerant to p-cresol than other C. difficile strains including 630, CF4 and CD196. Surprising, it was shown that Clostridium sordellii also has a high tolerance to p-cresol, suggesting an overlap in the tolerance pathways in these clostridial species.

Clostridium difficile is a leading cause of nosocomial infection in the developed world, causing antibiotic-associated disease in susceptible populations. The identity of immunogenic proteins is important in understanding the pathogenesis of disease and in the design of vaccines. This study analysed the sera of six patients infected during a hospital outbreak of a C. difficile ribotype 017 strain. Using a proteomics-based approach, cell wall proteins were separated by two-dimensional PAGE, and immunoreactive proteins were revealed by reaction with patient sera. The identity of immunoreactive proteins was established by MS. Forty-two different proteins were identified in total. All patient sera reacted with at least one component of the surface-layer protein (SLP), four reacted with both components (high- and low-molecular-mass SLPs), and five reacted with one other cell wall protein, suggesting that these are immunodominant antigens. The role of these proteins as potential vaccine candidates and their roles in pathogenesis deserve further study.

Clostridium difficile is a spore-forming anaerobic bacterium that is an emerging nosocomial threat; incidence of infection in hospitals is increasing, both in frequency and severity, resulting in considerable morbidity and mortality. In order to adapt to the intestinal environment, C. difficile must react to the many stresses involved with colonization, including exposure to antibiotics, which represents the most frequent precipitating agent of C. difficile infection. The responses of C. difficile to environmental shocks (heat, pH and oxidative shock) and to growth in the presence of subinhibitory concentrations of antibiotics (amoxicillin, clindamycin and metronidazole) were investigated using the C. difficile 630 microarray developed by the Bacterial Microarray Group at St George's, University of London, UK ( μG@S). Significantly regulated genes and operons were identified that are unique to or common between different stresses. The transcriptional profiles of C. difficile 630 are similar after growth in the presence of amoxicillin and clindamycin: both increased transcription of ribosomal protein genes and altered transcription of genes encoding surface-associated proteins. In contrast, metronidazole treatment resulted in minor changes in transcription patterns. The general stress response is observed after heat shock and acid shock. Heat shock also affected transcription of several biochemical pathways. Exposure to atmospheric oxygen induced a large number of electron transporters. This study provides a starting point for detailed analyses of numerous genes whose expression is affected by stress and may therefore be involved in adaptation to the host environment.

The intestinal epithelial cell line HT-29 was used to study the apoptotic effect of Clostridium difficile toxin A (TcdA). TcdA is a 300 kDa single-chain protein, which glucosylates and thereby inactivates small GTPases of the Rho family (Rho, Rac and Cdc42). The effect of TcdA-catalysed glucosylation of the Rho GTPases is well known: reorganization of the actin cytoskeleton with accompanying morphological changes in cells, leading to complete rounding of cells and destruction of the intestinal barrier function. Less is known about the mechanism by which apoptosis is induced in TcdA-treated cells. In this study, TcdA induced the activation of caspase-3, -8 and -9. Apoptosis, as estimated by the DNA content of cells, started as early as 24 h after the addition of TcdA. The impact of Rho glucosylation was obvious when mutant TcdA with reduced or deficient glucosyltransferase activity was applied. TcdA mutant W101A, with 50-fold reduced glucosyltransferase activity, induced apoptosis only at an equipotent concentration compared with wild-type TcdA at a 50 % effective concentration of 0.2 nM. The enzyme-deficient mutant TcdA D285/287N was not able to induce apoptosis. Apoptosis induced by TcdA strictly depended on the activation of caspases, and was completely blocked by the pan-caspase inhibitor z-VAD-fmk. Destruction of the actin cytoskeleton by latrunculin B was not sufficient to induce apoptosis, indicating that apoptosis induced by TcdA must be due to another mechanism. In summary, TcdA-induced apoptosis (cytotoxic effect) depends on the glucosylation of Rho GTPases, but is not triggered by destruction of the actin cytoskeleton (cytopathic effect).

The reported incidence and mortality of Clostridium difficile-associated disease has increased significantly, which in part is likely to be due to the emergence of a new, highly virulent strain in North America and Europe. This epidemic strain, referred to as BI/NAP1/027, has increased virulence, attributed to overexpression of the two toxin-encoding genes, tcdA and tcdB, which may be due to truncation of the negative regulator (tcdC) by a 1 bp deletion. In a previous study of whole-genome comparisons using microarray analysis of 75 C. difficile isolates, it was noted that the 20 027 strains, which formed a hypervirulent clade, possessed a unique hybridization pattern for the 7 toxin B microarray reporters. This unique pattern was conserved in all of these 027 strains. The pattern was different for the 55 non-027 strains tested. These data, along with the knowledge that 027 strains are toxinotype III (i.e. possess a complete tcdB gene of comparable size to toxin reference strain VPI 10463), suggest that the sequence of the N-terminal binding domain of toxin B must be divergent from C. difficile strain 630 (and the other 55 strains tested). Additionally, these 027 strains had comparable hybridization patterns across the whole microarray, as well as for tcdB. Therefore, it was suggested that they share a similar, novel N-terminal binding domain. The aim of this study was to ascertain the sequence variation in tcdB from eight characterized BI/NAP1/027 strains. The study confirmed significant sequence variation of tcdB from the sequenced strain 630 and slight variation in tcdB among the eight 027 strains. These results suggest that toxin B from 027 strains may have a different binding capacity compared with its less-virulent counterparts and may, in addition to the mutated tcdC regulator, be responsible for the increased virulence of 027 strains.

Clostridium difficile is the major cause of hospital-acquired infectious diarrhoea. Several antimicrobials are known to induce and promote C. difficile-associated diarrhoea (CDAD). The impact of metronidazole (MTR), vancomycin (VAN), clindamycin (CLI) and linezolid (LZD) on growth, toxin gene transcription and toxin production in C. difficile was investigated. Four C. difficile strains were grown with and without sub-MIC concentrations of MTR, VAN, CLI and LZD (0.5× MIC) and growth was measured by colony counts. Toxin production was detected using ELISA (for toxin A) and a cytotoxicity assay (for toxin B) in culture supernatants and also in sonicated cells. Real-time PCR was used to measure transcription of the toxin A and B genes. The aim of this work was to combine analysis of toxin A and B production by ELISA or cell culture assay with transcriptomic analysis. The four strains showed similar growth and different levels of toxin production in the absence of antibiotics. An antibiotic-free control showed toxin production at a late stage when the plateau phase of bacterial growth was reached, whereas antibiotic-exposed strains showed earlier toxin production. All of the antibiotics used except CLI increased the transcription rate of toxin genes. The findings of this study show that sub-MIC concentrations of antibiotics can cause changes in gene transcription of the major virulence factors of C. difficile. This study describes a new method for transcriptomic analysis of toxin genes in C. difficile.

The European Study Group on Clostridium difficile (ESGCD) conducted a prospective study in 2005 to monitor and characterize C. difficile strains circulating in European hospitals, collecting 411 isolates. Eighty-three of these isolates, showing resistance or intermediate resistance to moxifloxacin (MX), were selected for this study to assess susceptibility to other fluoroquinolones (FQs) and to analyse the gyr genes, encoding the DNA gyrase subunits GyrA and GyrB. Twenty MX-susceptible isolates from the surveillance study were included for comparison. Overall, one amino acid substitution in GyrA (Thr82 to Ile) and four different substitutions in GyrB (Ser416 to Ala, Asp426 to Asn, Asp426 to Val and Arg447 to Lys) were identified. A high level of resistance (MIC ≥32 μg ml−1) to MX, ciprofloxacin (CI), gatifloxacin (GA) and levofloxacin (LE) was found in 68 isolates showing the amino acid substitution Thr82 to Ile in GyrA, in eight isolates with the substitutions Thr82 to Ile in GyrA and Ser416 to Ala in GyrB, in two isolates showing the substitution Asp426 to Asn in GyrB and in one isolate with Asp426 to Val in GyrB. The remaining four isolates showed high MICs for CI and LE, but different MIC levels for MX and GA. In particular, intermediate levels of resistance to MX were shown by two isolates, one with the substitution Thr82 to Ile in GyrA, and one showing Asp426 to Asn in GyrB. The substitution Arg447 to Lys in GyrB was found in two strains resistant to MX, CI and LE but susceptible to GA. No substitutions in GyrA were found in the FQ-susceptible strains, whereas two strains showed the amino acid change Ser416 to Ala in GyrB. Thr82 to Ile was the most frequent amino acid change identified in the C. difficile isolates examined. In contrast to previous observations, 10 % of the isolates showed this substitution in association with Ser416 to Ala in GyrB. The other amino acid changes found were characteristic of a few strains belonging to certain types and/or countries. Two new substitutions for C. difficile, Ser416 to Ala and Arg447 to Lys, were found in GyrB. Whereas the former does not seem to have a key role in resistance, since it was also detected in susceptible strains, the latter substitution occurred in the same position where other amino acid variations take place in resistant Escherichia coli and other C. difficile strains. A large number of C. difficile isolates now show an alarming pattern of resistance to the majority of FQs currently used in hospitals and outpatient settings, therefore judicious use of these antibiotics and continuous monitoring of in vitro resistance are necessary.

A total of 313 faecal samples from three pig farms and two cattle farms was collected, and Clostridium difficile was isolated from 133/257 piglet samples (51.8 %) and from 1/56 calf samples (1.8 %). Toxins were tested only in calf samples and were positive in 44.6 % (25/56). The only bovine isolate belonged to toxinotype XIa (A−B−CDT+). Porcine isolates belonged to toxinotype 0 (A+B+CDT−) and toxinotype V (A+B+CDT+). Of the two ribotypes usually found in toxinotype V, the strains isolated in this study showed a greater similarity to ribotype 066 than to ribotype 078.