- Volume 60, Issue 8, 2011

Clostridial glucosylating toxins are the main virulence factors of clostridia responsible for gangrene and/or colitis. These toxins have been well characterized to inactivate Rho/Ras-GTPases through glucosylation. However, the signalling pathways downstream of Rho/Ras-GTPases leading to the intracellular effects of these toxins are only partially known. Rac-dependent modification of focal adhesion complexes and phosphoinositide metabolism seem to be key processes involved in actin filament depolymerization and disorganization of intercellular junctions. In addition, clostridial glucosylating toxins induce Rho/Ras-independent intracellular effects such as activation of mitogen-activated protein kinase pathways, which are used by some of these toxins to trigger an inflammatory response.

Clostridium difficile is the most common cause of nosocomial bacterial diarrhoea in the Western world. Diarrhoea and colitis are caused by the actions of toxins A and B released by pathogenic strains of C. difficile. Adaptive immune responses to these toxins influence the outcomes of C. difficile infection (CDI). Symptomless carriers of toxinogenic C. difficile and those with a single episode of CDI without recurrence show more robust antitoxin immune responses than those with symptomatic and recurrent disease. Immune-based approaches to CDI therapy and prevention have been developed using active vaccination or passive immunotherapy targeting C. difficile toxins. Innate immune responses to C. difficile and its toxins are also central to the pathophysiology of CDI. An acute intestinal inflammatory response with prominent neutrophil infiltration and associated tissue injury is characteristic of CDI. Furthermore, inhibiting this acute inflammatory response can protect against the intestinal injury that results from exposure to C. difficile toxins in animal models. Studies examining host risk factors for CDI have led to validated clinical prediction tools for risk of primary and of recurrent disease. Risk factors associated with severe CDI with poor clinical outcomes have also been identified and include marked elevation of the peripheral white blood cell count and elevated creatinine. However, further work is needed in this area to guide the clinical application of new approaches to disease prevention and treatment including new antimicrobials as well as passive and active immunization.

A repetitive-extragenic palindromic PCR (rep-PCR) subtyping method (DiversiLab) in conjunction with ribotyping, toxinotyping and antimicrobial-susceptibility testing was used to detect subtypes within Clostridium difficile ribotypes 027 and 078. Clinical isolates of ribotypes 027 (toxinotype III) (n = 30) and 078 (toxinotype V) (n = 23) were provided by health-care facilities across the Republic of Ireland over 2 months in 2006 and 1 month in 2009. Ribotype 027 isolates were significantly more related to each other (9 different subtype profiles) when compared to ribotype 078 isolates (14 different profiles) (P = 0.001; cut-off >90 % similarity). Almost half of ribotype 078 isolates (45.5 %) showed no relationship to each other. The clonality of ribotype 027 isolates suggests effective adaptation to the human niche, whereas the considerable genetic diversity within ribotype 078 isolates suggests that they may have originated from a variety of sources. Subtyping correlated well with antimicrobial susceptibility, in particular clindamycin susceptibility for ribotype 027, but diverse antimicrobial-susceptibility profiles were seen in ribotype 078 isolates, even within a single health-care facility. Between 2006 and 2009, a change in the predominant subtype of ribotype 027 was seen, with the recent clone representing half of all ribotype 027 isolates studied. This strain exhibited 89 % similarity to a rep-PCR profile of the North American NAP-1 strain.

The objective of this study was to evaluate the usefulness of multilocus variable-number tandem repeat analysis (MLVA) for typing and subtyping of Clostridium difficile. Sixty-eight strains were studied, including strains from PCR ribotypes 027, 078/126, 014/020/077, 017 and 023. The stability of variable-number tandem repeat (VNTR) loci was tested by comparing the MLVA results of two strains subcultured 11 times. After DNA extraction, seven tandem repeat loci (A6, B7, C6, E7, F3, G8, H9) from published MLVA schemes were amplified by PCR and sequenced. The distance between two strains was determined by calculating the summed tandem repeat difference. Genomic diversity was evaluated by using the minimum spanning tree (Bionumerics 5.1 software program; Applied Maths). Among the 68 C. difficile isolates examined, 65 unique MLVA types were identified, suggesting a high discriminatory power. An overall good agreement was observed between MLVA types and PCR ribotypes. The stability of VNTR loci was good. MLVA could separate isolates of the hypervirulent PCR ribotype 027 clone in several clusters; all 027 strains isolated within a hospital were grouped in a specific cluster or were placed very close to each other. Results of MLVA confirmed that strains from PCR ribotypes 078 and 126 were closely related although some were located in different branches of the tree. Similar results were observed for most strains from PCR ribotypes 014, 020 and 077. This highly discriminatory method is time-consuming and expensive, but is a valuable tool for subtyping of C. difficile, especially of 027 strains.

PFGE is currently the North American standard for surveillance for Clostridium difficile but lacks discriminatory power to aid outbreak investigation. A further limitation to PFGE is the high baseline rate of the epidemic North American pulsotype (NAP) 1 strain in hospitals. Multilocus variable-number tandem repeat analysis (MLVA) appears to have superior discriminatory power but criteria to define clonality have not been set. We conducted surveillance for toxin-positive C. difficile infection (CDI) at a single academic health sciences centre between September 2009 and April 2010. Seventy-four patient specimens resulting in 86 discrete CDI episodes were subjected to PFGE and MLVA. Results were analysed using Bionumerics software to generate phylogenetic trees and coupled to patient demographic data. Amongst the NAP1 strains, two distinct clusters were identified by MLVA using 90 % similarity as a cut-off by Manhattan distance-based clustering, four clusters using 95 % and seven clusters using 97 %. Population analysis conducted on multiple colonies (n = 25) demonstrated that 1–3 % difference in MLVA types was typical for a single individual. Typing was also conducted in the context of institutional outbreaks (n = 42, three outbreaks) in order to determine clusters within the NAP1 strain. By combining longitudinal surveillance with epidemiological information, single specimen population analysis and typing in the context of institutional outbreaks, we conclude that the use of the Manhattan distance-based clustering with a cut-off of 95–97 % is capable of distinguishing outbreak clones from sporadic isolates.

Clostridium difficile strains of toxinotypes III (n = 13) and V (n = 45) were typed by agarose gel-based PCR ribotyping, capillary gel electrophoresis-based PCR ribotyping and PFGE using two different restriction enzymes, SmaI and SacII. With conventional agarose gel-based PCR ribotyping, toxinotype III strains were distributed among six different PCR ribotypes and toxinotype V strains into three different PCR ribotypes. Capillary gel electrophoresis-based ribotyping was more discriminatory for toxinotype V strains, with six different ribotypes found. With PFGE using SmaI, all toxinotype III strains grouped together into a single pulsotype. Using SacII, ribotype 027 strains grouped together with >90 % similarity and were <83 % similar to other ribotypes of toxinotype III strains. Within ribotype 078, seven (SmaI) and eight (SacII) different pulsotypes were found, whilst ribotype 126 strains belonged to one (SmaI) and two (SacII) pulsotypes. Within ribotype 066, it was possible to distinguish between pig and human isolates. Using SacII, a further distinction could also be made between pig isolates from two different farms. PFGE (SmaI and SacII) clustered strains according to their toxinotype; however, correlation of PFGE and ribotyping was better with SacII. These data suggest that toxinotype III strains are a more heterogeneous group than toxinotype V strains and that SacII is more discriminatory than SmaI. Alternatively, the use of both enzymes simultaneously could improve PFGE typing of C. difficile.

A total of 817 human clinical isolates of Clostridium difficile from all Australian states were screened for A−B+ strains by toxin gene PCR assays. Nine (1.1 %) strains were confirmed to be A−B+ by enzyme immunoassay for toxin production. Of these, six (66.7 %) were binary toxin-positive by PCR. Using PCR ribotyping and toxinotyping, the A−B+ strains could be grouped into seven ribotypes and three toxinotypes. Only one of the ribotypes had been reported previously (017). The prevalence of ribotype 017 was low in this study with only two strains detected. Two new A−B+ toxinotypes were also defined (XXX, XXXI). Toxinotype XXX had a toxin B gene similar to that of toxinotype IV (A+B+) but with a novel cytopathic region. Toxinotype XXXI was similar to other A−B+ types (X, XVII), but had a larger deletion to the toxin A gene than in either of those types. The types of A−B+ strains identified in this study differed markedly from those described in other regions.

During early infancy asymptomatic intestinal colonization by Clostridium difficile is frequent. To update information on infant colonization prevalence and to characterize infant strains, in terms of their virulence factors and their phylogenetic diversity, a prospective screening of C. difficile in the stools of infants 0 to 2 years old was conducted at Jean Verdier Hospital (Hôpital Jean Verdier) over an 18 month period. C. difficile was screened by toxigenic culture, and molecular characterization was performed by PCR-ribotyping and multilocus sequence typing (MLST). The overall C. difficile colonization prevalence was 33.7 % (99/294). The colonization rate by a toxigenic strain was 7.1 % (21/294). Community-acquired C. difficile accounted for 66.7 % (66/99) of cases. Molecular typing was performed on 90 isolates from Jean Verdier Hospital and 8 additional isolates from another hospital in Versailles (Centre Hospitalier de Versailles). Among these isolates, 23 were toxigenic (21 tcdA+/tcdB + and 2 tcdA−/tcdB +). All the isolates were negative for the binary toxin genes. Seventeen PCR ribotypes (PRs) were identified, with five PRs accounting for 82.7 % (81/98) of the isolates. MLST generated 15 different sequence types (STs). The predominant genotype, PRJV11-ST38 (33.7 %), included only non-toxigenic strains. Toxigenic strains were distributed in eight genotypes. Neither PR027-ST3, nor PR078/126-ST49 were identified but some PRs/STs corresponded to well-known adult infectious strains. These results indicate that infants are widely colonized by non-toxigenic strains. However, toxigenic adult infectious strains circulate in asymptomatic infants even in the community; thus, infants may be a reservoir for adult infectious strains.

Clostridium difficile has emerged as a pathogen or commensal in food animals. There is overlap between isolates from animals, retail meats and humans, suggesting that animals may be a C. difficile reservoir. For direct detection of variant C. difficile strains in faecal samples of symptomatic and asymptomatic animals, we developed and validated a new TaqMan real-time PCR (TMrtPCR) assay targeting the tcdA, tcdB and cdtB genes. We compared it with the enrichment culture method and with two real-time PCR (rtPCR) assays, BrtPCR and PCRFast, targeting tcdB and tcdA/tcdB, respectively. All ten tested C. difficile toxinotypes, except one (XIa) with PCRFast and two (X, XIa) with BrtPCR, were detected with the test assays. A total of 340 (100 %) samples were cultured and amplified with TMrtPCR. Results correlated in 75.3 % samples. Forty (11.8 %) samples were culture positive/TMrtPCR negative, possibly because of the low numbers of bacteria in the samples or because of DNA extraction failure. Forty (11.8 %) samples were TMrtPCR positive/culture negative. Among 79 samples included in the rtPCR assays/culture comparison, 50.6 % were in complete concordance. The results showed that TMrtPCR performed better than BrtPCR and PCRFast, and 67 % of the culture-positive samples were TMrtPCR positive in comparison to 40 % of the samples positive in BrtPCR and 7 % of the samples positive in PCRFast, respectively. Another advantage of TMrtPCR over BrtPCR and PCRFast is its ability to detect a binary toxin gene. Therefore, the TMrtPCR results can provide the first information about the toxin type present in the sample. According to the results of our study, TMrtPCR could be a preferred screening method for the rapid detection of C. difficile in animal faecal samples, although an enrichment culture has to be performed for the specimens with negative or inconclusive rtPCR results.

The loop-mediated isothermal amplification (LAMP) assay detecting the slpA gene of slpA sequence type gc8 (slpA-gc8) was established for the identification of a hypervirulent Clostridium difficile strain, PCR ribotype 027. Of 107 isolates examined, 27 belonging to PCR ribotype 027 were all positive for the LAMP assay. The remaining 80 isolates were typed into 47 different PCR ribotypes other than type 027, and were negative for the LAMP assay with the exception of two isolates. The sensitivity and specificity of the LAMP method for identification of PCR ribotype 027 were 100 % and 98 %, respectively. The LAMP assay detecting slpA-gc8 is a reliable tool for the identification of PCR ribotype 027 C. difficile. This simple and rapid method will contribute to detection of the hypervirulent strain.

Two commercial real-time PCR assays for the detection of Clostridium difficile, BD GeneOhm Cdiff assay (BD Diagnostics) and Xpert C. difficile assay (Cepheid), were compared to each other and to toxigenic culture, which was used as a gold standard, on a set of 194 clinical stools submitted for routine diagnostic analysis. Of 28 (14.4 %) toxigenic culture positive samples 23 were positive with both assays, the BD and the Cepheid real-time PCR assays, 4 were positive only by Cepheid Xpert C. difficile assay and 1 sample was negative by both PCR assays, resulting in sensitivity, specificity, positive predictive value and negative predictive value of 82.1, 98.2, 88.5 and 97.0 %, respectively, for the BD GeneOhm Cdiff assay, and 96.4, 97.3, 87.1 and 99.3 %, respectively, for the Cepheid Xpert C. difficile assay. Altogether 26 out of 194 (13.4 %) samples were reported invalid by Cepheid. Toxigenic C. difficile positive samples contained 15 different PCR ribotypes distributed into toxinotype 0 and 2 different variant toxinotypes (III, IV). Clinical data were available for 24 out of 28 (85.7  %) toxigenic C. difficile positive patients and 18 (75.0 %) of them were diagnosed with diarrhoea, while others had other symptoms or risk factors related to possible C. difficile infection (antibiotics, bloody stool, peritonitis, Crohn’s disease).

The objective of this study was to compare full binary toxin loci (CDTloc) sequences from a collection of Clostridium difficile isolates in an effort to further understand the regulation of the binary toxin (CdtAB) and its putative regulator (CdtR). Sequences from different ribotypes and toxinotypes were analysed phylogenetically and for polymorphisms, non-sense mutations, promoter features and signal sequences. Expression of cdtA, which was also representative of cdtB expression, was measured by quantitative PCR (qPCR). Several consensus promoter features and various polymorphisms were identified including a non-sense mutation identified in a ribotype 078 cdtR gene that is predicted to result in a severely truncated protein. Despite this mutation, cdtA expression was still detected by qPCR. Dendrograms based on total sequences indicated that isolates belonging to the same ribotype shared the greatest similarity within the binary toxin locus. Although cdtR is thought to be involved in regulation of cdtA expression, a cdtR non-sense mutation did not inhibit expression of cdtA, suggesting that either the truncated protein is functional or another regulator of the binary toxin exists.

Clostridium difficile is the major cause of antibiotic-associated colitis, a disease with significant morbidity and mortality. This study investigated the role of the haem oxygenase-1 (HO-1)/carbon monoxide (CO) pathway in C. difficile toxin A-induced enteritis in mice. The HO substrate haemin, zinc protoporphyrin IX (ZnPP IX), a specific HO-1 inhibitor, dimanganese decacarbonyl (DMDC), a CO donor, or an equivalent volume of their respective vehicles were injected subcutaneously 30 min prior to local challenge with toxin A (25 or 50 µg per ileal loop) or PBS. Intestinal ileal loop weight/length ratios were calculated 3 h later. Ileal tissues were collected for histological analysis and measurement of myeloperoxidase (MPO) activity, tumour necrosis factor alpha (TNF-α) and interleukin-1 beta (IL-1β) production by ELISA and immunohistochemistry for HO-1. Treatment of mice subjected to C. difficile toxin A (TcdA) with haemin or DMDC prevented oedema, mucosal disruption and neutrophil infiltration observed in histological analysis. It also decreased TcdA-induced MPO activity and TNF-α or IL-1β production. In contrast, the specific HO-1 inhibitor (ZnPP IX) exacerbated all these evaluated parameters. TcdA increased HO-1 expression as seen by immunohistochemistry. These results suggest that the HO-1/CO pathway exerts a protective role in TcdA-induced enteritis and that its pharmacological modulation might be important for the management of C. difficile-associated disease.

Clostridium difficile is a frequent cause of severe, recurrent, post-antibiotic diarrhoea and pseudomembranous colitis. Its pathogenicity is mediated mainly by two toxins, TcdA and TcdB. However, different adhesins have also been described as important colonization factors which are implicated in the first step of the intestinal infection. In this study, we focused our interest on one of these adhesins, fibronectin-binding protein A (FbpA), and on its role in the intestinal colonization process. A mutant of FbpA (CDΔFbpA) was constructed in C. difficile strain 630Δerm by using ClosTron technology. This mutant was characterized in vitro and in vivo and compared to the isogenic wild-type strain. Adhesion of the CDΔFbpA mutant to the human colonic epithelial cell line Caco-2 and to mucus-secreting HT29-MTX cells was examined. Surprisingly, the CDΔFbpA mutant adhered more than the wild-type parental strain. The CDΔFbpA mutant was also analysed in three different mouse models by following the intestinal implantation kinetics (faecal shedding) and caecal colonization (7 days post-challenge). We showed that in monoxenic mice, CDΔFbpA shed C. difficile in faeces at the same rate as that of the isogenic wild-type strain but its colonization of the caecal wall was significantly reduced. In dixenic mice, the shedding rate was slower for the CDΔFbpA mutant than for the isogenic wild-type strain during the first days of infection, but no significant difference was observed in caecal colonization. Similar rates of intestinal implantation and caecal colonization were observed for both strains in assays performed in human microbiota-associated mice. Taken together, our data suggest that FbpA plays a role in intestinal colonization by C. difficile.

Surface-layer proteins (SLPs) have been detected in all Clostridium difficile strains and play a role in adhesion, although an involvement in the inflammatory process may also be supposed, as they cover the bacterial surface and are immunodominant antigens. The aim of this study was to evaluate the immunomodulatory properties of SLPs obtained from hypervirulent and epidemic (H/E) or non-H/E C. difficile strains, to try to determine whether they contribute to hypervirulence. SLPs were purified from H/E PCR ribotype 027 and 001 and non-H/E PCR ribotype 012 C. difficile strains, and the ability to modulate these properties was studied in human ex vivo models of monocytes and monocyte-derived dendritic cells (MDDCs). The results indicated that SLPs were able to induce immunomodulatory cytokines [interleukin (IL)-1β, IL-6 and IL-10] in monocytes. SLPs induced maturation of MDDCs, which acquired enhanced antigen-presenting activity, a crucial function of the mature stage. SLP-primed MDDCs expressed high levels of IL-10, an important regulatory cytokine. No significant differences were found in the activation induced in monocytes and MDDCs by SLP preparations from H/E and non-H/E strains. Overall, these findings show an important role for SLPs in modulation of the immune response to C. difficile. However, SLPs from H/E strains did not show a specific immunomodulatory pattern compared with SLPs from non-H/E strains, suggesting that SLPs are not involved in the increased severity of infection peculiar to H/E strains.

The aim of this study was to investigate the S-layer proteins (SLPs) of the hypervirulent Clostridium difficile PCR ribotype 027 and compare them with those of PCR ribotype 001 and other PCR ribotypes involved in C. difficile infection and outbreaks, by molecular analysis and immunological assays. It has been demonstrated previously that PCR ribotype 027 SlpA is conserved in C. difficile strains belonging to this PCR ribotype and that it is a new variant, showing 88 % identity with SlpA of PCR ribotype 001. As the low-molecular-weight (LMW) SLPs of C. difficile are immunodominant antigens, attention was focused on this region of the genome. Sequencing of strains of different PCR ribotypes (001, 012, 014, 017, 027 and 078) showed that SlpA was conserved among strains belonging to the same PCR ribotype. Comparison of the LMW SLP region among these strains identified ten regions with sequence identity between PCR ribotypes 027 and 001, and low conservation with the other PCR ribotypes. In particular, two of these regions corresponded to areas predicted to be surface exposed. Three specific peptides, including those of the two surface-exposed regions, were recognized by human sera against PCR ribotypes 027 and 001 and by a rabbit polyclonal serum against the SLPs of PCR ribotype 027. In contrast, these peptides were not recognized by a polyclonal serum against the SLPs of PCR ribotype 012 used as a control. These results confirm the antigenic role of the LMW SLP and suggest that it may have a role in evasion of the host immune response.

Clostridium difficile is the main cause of antibiotic-associated disease, a disease of high socio-economical importance that has recently been compounded by the global spread of the 027 (BI/NAP1/027) ribotype. C. difficile cases attributed to ribotype 027 strains have high recurrence rates (up to 36 %) and increased disease severity. The hamster model of infection is widely accepted as an appropriate model for studying aspects of C. difficile host–pathogen interactions. Using this model we characterized the infection kinetics of the UK 2006 outbreak strain, R20291. Hamsters were orally given a dose of clindamycin, followed 5 days later with 10 000 C. difficile spores. All 100 % of the hamsters succumbed to infection with a mean time to the clinical end point of 46.7 h. Colonization of the caecum and colon were observed 12 h post-infection reaching a maximum of approximately 3×104 c.f.u. per organ, but spores were not detected until 24 h post-infection. At 36 h post-infection C. difficile numbers increased significantly to approximately 6×107 c.f.u. per organ where numbers remained high until the clinical end point. Increasing levels of in vivo toxin production coincided with increases in C. difficile numbers in organs reaching a maximum at 36 h post-infection in the caecum. Epithelial destruction and polymorphonuclear leukocyte (PMN) recruitment occurred early on during infection (24 h) accumulating as gross microvilli damage, luminal PMN influx, and blood associated with mucosal muscle and microvilli. These data describe the fatal infection kinetics of the clinical UK epidemic C. difficile strain R20291 in the hamster infection model.

Herein we present evidence for the therapeutic potential of colonization factor (CF)-specific egg yolk antibodies (IgY) for potentially treating acute and recurring Clostridium difficile infection (CDI) in humans. The study involved cloning, expressing as 6×His-tagged proteins in Escherichia coli, and Ni-affinity purifying three previously identified CFs (FliC, FliD and Cwp84) from C. difficile. The recombinant CF antigens were then used to immunize Leghorn chickens and CF-specific IgY antibodies were prepared from their eggs. The specificity and titre of the resulting C. difficile CF-specific IgY antibodies were assessed by ELISA and Western immunoblotting techniques. The antibodies were also screened for their ability to inhibit C. difficile adherence to human colon-derived T84 cells, and, based on these findings, one of them (FliD-specific IgY) was evaluated for its potential to prevent C. difficile-mediated morbidity and mortality in Syrian hamsters. The results revealed that purified FliD-specific IgY significantly protected hamsters from C. difficile strain 630 infection relative to control animals treated with carbonate buffer alone or IgY produced from unimmunized chicken eggs. The results suggest that egg yolk preparations obtained from chickens immunized with recombinant C. difficile CFs may represent another safe and cost-effective treatment option in humans suffering from acute or recurring CDI.

During a 24 month period from 2007 to 2009, 174 faecal specimens from horses in Australia (predominantly from Western Australia) were tested for Clostridium difficile. C. difficile was isolated from 14 (23 %) of 62 diarrhoeal animals (including 10 foals) and from none of 112 healthy adult horses. These isolates were toxin profiled by PCR for toxin A, toxin B and binary toxin, and ribotyped. Ten of the equine isolates were A+B+CDT−. Other toxin profiles detected were A−B−CDT+ (one isolate), A+B+CDT+ (two isolates) and A−B−CDT− (three isolates). There were six different ribotypes detected in the horses, ribotype 012 being the most common with six isolates. Two horses (one adult and one foal) had two strains of C. difficile isolated on different days. These strains had the same toxin profile but different ribotypes. None of the equine isolates was ribotype 078, which is A+B+CDT+ and a significant cause of animal disease overseas. All isolates were susceptible to metronidazole and vancomycin. These results suggest that the epidemiology of C. difficile in horses in Australia is currently similar to that in other parts of the world, but requires further surveillance to monitor changes.