- Volume 62, Issue 9, 2013

Clostridium difficile is a frequent cause of severe, recurrent post-antibiotic diarrhoea and pseudomembranous colitis. The surface layer (S-layer) is the predominant outer surface component of C. difficile which is involved in pathogen–host interactions critical to pathogenesis. In this study, we characterized the S-layer protein A (SlpA) of animal and human strains belonging to different PCR-ribotypes (PR) and compared the in vitro adherence and in vivo colonization properties of strains showing different SlpA variants. Since each SlpA variant has been recently associated with an S-layer cassette, we were able to deduce the cassette for each of our strains. In this study, an identity of 99–100 % was found among the SlpA of isolates belonging to PR 012, 014/020, 045 and 078. One exception was the SlpA of a poultry isolate, PR 014/020, which showed 99 % identity with that of strain 0160, another PR 014/020 which contains an S-layer cassette 6. Interestingly, this cassette has also been found in a PR 018 strain, an emerging virulent type currently predominant in Italy. Five other SlpA variants (v014/020a–e) were identified in strains PR 014/020. In vitro adherence assays and in vivo colonization experiments were performed on five PR 014/020 strains: human 1064 (v014/020e), human 4684/08 (v014/020b), human IT1106 (v078a), poultry P30 (v014/020d) and poultry PB90 (v014/020b) strains. Adhesion assays indicate that C. difficile strains vary in their capacity to adhere to cells in culture and that adhesion seems to be independent of the SlpA variant. Colonization properties were assessed in vivo using a dixenic mouse model of colonization. The kinetics of faecal shedding and caecal colonization were similar when human 4684/08 (v014/020b) strain was compared with human 1064 (v014/020e) and poultry PB90 (v014/020b) strain. In contrast, poultry P30 (v014/020d) strain outcompeted both human 4684/08 (v014/020b) and IT1106 (v078a) strains and its adherence to caeca at day 7 was significantly higher. The peculiar characteristics of C. difficile P30 seem to advantage it in colonizing the intestinal mice niche, increasing its ability to compete and adapt. The results obtained underline the need of an increased attention to the genetic evolution of C. difficile to prevent and limit the consequences of the emergence of increasingly virulent strains.

Clostridium difficile infection (CDI) has been identified as the leading cause of nosocomial diarrhoea and pseudomembranous colitis associated with antibiotic therapy. Recent epidemiological changes as well as increases in the number of outbreaks of strains associated with increased virulence and higher mortality rates underscore the importance of identifying alternatives to antibiotics to manage this important disease. Animal studies have clearly demonstrated the roles that toxins A and B play in gut inflammation as well as diarrhoea; therefore it is not surprising that serum anti-toxin A and B IgG are associated with protection against recurrent CDI. In humans, strong humoral toxin-specific immune responses elicited by natural C. difficile infection is associated with recovery and lack of disease recurrence, whereas insufficient humoral responses are associated with recurrent CDI. The first generation of C. difficile vaccine that contained inactivated toxin A and B was found to be completely protective against death and diarrhoea in the hamster C. difficile challenge model. When tested in young healthy volunteers in Phase I clinical trials, this investigational vaccine was shown to be safe and immunogenic. Moreover, in a separate study this vaccine was able to prevent further relapses in three out of three patients who had previously suffered from chronic relapsing C. difficile-associated diarrhoea. Herein we examined the immunogenicity and protective activity of a next-generation Sanofi Pasteur two-component highly purified toxoid vaccine in a C. difficile hamster model. This model is widely recognized as a stringent and relevant choice for the evaluation of novel treatment strategies against C. difficile and was used in preclinical testing of the first-generation vaccine candidate. Intramuscular (i.m.) immunizations with increasing doses of this adjuvanted toxoid vaccine protected hamsters from mortality and disease symptoms in a dose-dependent manner. ELISA measurements of pre-challenge sera showed that the median anti-toxin A and anti-toxin B IgG titres in the group of surviving animals were significantly higher than the median values in the group of animals that did not survive challenge. Assessment of the neutralizing activity of these sera revealed a statistically significant difference between the levels of both toxin A and toxin B neutralizing titres in protected versus unprotected animals as the median anti-toxin A and anti-toxin B neutralizing titres from surviving animals were higher than the median values from animals that succumbed to challenge. Statistically significant correlations between the toxin-specific binding titres and toxin neutralizing titres were seen for both toxin A and toxin B responses. The role of circulating anti-toxin antibodies in immunity against disease was evaluated by passive transfer of immune sera against C. difficile toxoids to naïve hamsters. Passively immunized animals were protected against morbidity and mortality associated with C. difficile challenge. Taken together, these results indicate the ability of i.m. immunization with inactivated toxins A and B to induce robust dose-dependent anti-toxin A and anti-toxin B IgG responses, the principal role of circulating anti-toxin antibody in immunity against disease and that antibody toxin binding and neutralization titres can serve as correlates of protection in the hamster challenge model of C. difficile.

Spore germination is an important part of the pathogenesis of Clostridium difficile infection (CDI). Spores are resistant to antibiotics, including those therapeutically administered for CDI and strains with a high germination rate are significantly more likely to be implicated in recurrent CDI. The role of germination efficiency in cases of refractory CDI where first-line therapy fails remains unclear. We investigated spore germination efficiencies of clinical C. difficile isolates by measuring drop in OD600 and colony forming efficiency. Ribotype 027 isolates exhibited significantly higher germination efficiencies in the presence of 0.1 % (w/v) sodium taurocholate (51.66±8.75 %; 95 % confidence interval (CI) 47.37–55.95 %) than ribotype 106 (41.91±8.35 %; 95 % CI 37.82–46 %) (P<0.05) and ribotype 078 (42.07±8.57 %, 95 % CI 37.22–46.92 %) (P<0.05). Spore outgrowth rates were comparable between the ribotype groups but the exponential phase occurred approximately 4 h later in the absence of sodium taurocholate. Spore germination efficiencies for isolates implicated in severe CDI were significantly higher (49.68±10.00 %, 95 % CI 47.06–52.30 %) than non-severe CDI (40.92±9.29 %, 95 % CI 37.48–44.36 %); P<0.01. Germination efficiencies were also significantly higher in recurrent CDI or when metronidazole therapy failed than when therapy was successful [(49.00±10.49 %, 95 % CI 46.25–51.75 %) versus (41.42±9.43 %, 95 % CI 37.93–44.91 %); P<0.01]. This study suggests an important link between C. difficile spore germination, CDI pathogenesis and response to treatment; however, further work is warranted before the complex interplay between germination dynamics and CDI outcome can be fully understood.

The combined repetitive oligopeptides (CROPs) of Clostridium difficile toxins A (TcdA) and B (TcdB) induce clathrin-mediated endocytosis of the toxins. Inconsistently, CROP-truncated TcdA1–1874 is also capable of entering host cells and displaying full cytotoxic properties although with less potency. Pre-incubation of cells with isolated CROPs, however, reconstitutes the reduced uptake of TcdA1–1874 to the level of the full-length toxin. We believe that TcdA exhibits an additional binding motif beyond the C-terminally located CROP domain, which might interact with cellular receptor structures that are associated with alternative internalization pathways. This study therefore evaluated endocytosis routes of CROP-dependent cellular uptake for TcdA and CROP-independent cellular uptake for TcdA1–1874. Clathrin knockdown or inhibition with chlorpromazine affected subsequent internalization of TcdA and TcdA1–1874, although only to some extent, arguing for alternative, clathrin-independent endocytosis routes. Inhibition of dynamin, a GTPase essentially involved in clathrin-mediated endocytosis as well as in various clathrin-independent uptake mechanisms, affected uptake of TcdA to the same extent as clathrin inhibition. In contrast, uptake of TcdA1–1874 was almost completely eliminated in dynamin-inhibited cells. Thus, clathrin-independent uptake of TcdA1–1874 presumably depends on dynamin. These findings demonstrate that the toxins are endocytosed via complex pathways involving clathrin and dynamin, putatively enabling them to adapt to mechanisms of various cell types. With regard to the emergence of C. difficile strains producing C-terminally truncated toxins, this study emphasizes the relevance of elucidating toxin uptake as a prerequisite for the development of toxin intervention strategies.

The culture of toxigenic Clostridium difficile from stool specimens is still seen as the gold standard for the laboratory diagnosis of C. difficile infection (CDI). bioMérieux have released ChromID Cdiff chromogenic agar (CDIF) for the isolation and identification of C. difficile in 24 h. In this study, we compared CDIF to pre-reduced cycloserine-cefoxitin-fructose agar with sodium taurocholate (TCCFA) in the examination of glutamate dehydrogenase-positive faecal specimens that were either GeneOhm positive or negative, using direct culture or culture following alcohol shock. Direct culture on CDIF had a sensitivity of 100 % and recovery of 94 % while for TCCFA these were 87 % and 82 %, respectively. For GeneOhm-positive alcohol-shocked faecal samples, sensitivity and recovery on CDIF was similar to direct culture while on TCCFA they were about 10 % higher. For direct culture, there was a significant difference between growth on CDIF at 24 h and TCCFA at 48 h (P = 0.001) and between the two media at 48 h (P<0.001). A total of 142 strains of C. difficile were recovered in pure culture from all GeneOhm-positive samples used in this study and 11 (7.7 %) of these were A−B−CDT− and may represent mixed infections of toxigenic and non-toxigenic C. difficile. The most dominant ribotype was UK 014 (14.7 %) followed by 002 (11.9 %) and 020 (11.9 %), and 36 % of toxigenic isolates, including an A−B+CDT− strain, could not be assigned a UK ribotype. CDIF outperformed pre-reduced TCCFA by negating the need for alcohol shock treatment and by giving a time saving of 24 h in the isolation of C. difficile. CDIF plates were also more selective than TCCFA and C. difficile colonies were easy to identify and subculture prior to strain typing.

The increasing incidence of Clostridium difficile infection (CDI) in Bulgaria has indicated the need to implement better surveillance approaches. The aim of the present work was to improve the current surveillance of CDI in Bulgaria by introducing innovative methods for identification and typing. One hundred and twenty stool samples obtained from 108 patients were studied over 4 years from which 32 C. difficile isolates were obtained. An innovative duplex EvaGreen real-time PCR assay based on simultaneous detection of the gluD and tcdB genes was developed for rapid C. difficile identification. Four toxigenic profiles were distinguished by PCR: A+B+CDT− (53.1 %, 17/32), A−B+CDT− (28.1 %, 9/32), A+B+CDT+ (9.4 %, 3/32) and A−B−CDT− (9.4 %, 3/32). PCR ribotyping and multilocus variable number of tandem repeat analysis (MLVA7) were used for molecular characterization of the isolates. In total, nine distinct ribotypes were confirmed and the most prevalent for Bulgarian hospitals was 017 followed by 014/020, together accounting for 44 % of all isolates. Eighteen per cent of the isolates (6/32) did not match any of the 25 reference ribotypes available in this study. Twenty-four MLVA7 genotypes were detected among the clinical C. difficile isolates, distributed as follows: five for 017 ribotype, two for 014/020, 001, 002, 012 and 046 each, and one each for ribotypes 023, 070 and 078. The correlation between the typing methods was significant and allowed the identification of several clonal complexes. These results suggest that most C. difficile cases in the eight Bulgarian hospitals studied were associated with isolates belonging to the outbreak ribotypes 017 and 014/20, which are widely distributed in Europe. The real-time PCR protocol for simultaneous detection of gluD and tcdB proved to be very effective and improved C. difficile identification and confirmation of clinical C. difficile isolates.

The presence of Clostridium difficile in food like shellfish, vegetables and meat has been reported in several publications during the past few years. The objective of this study was to assess the prevalence of ready-to-eat raw vegetables contaminated with C. difficile in France. One hundred and four ready-to-eat salads and vegetables were studied. Toxigenic C. difficile strains were isolated in three samples (2.9 %): two ready-to-eat salads (one heart of lettuce and one lamb’s lettuce salad) and one portion of pea sprouts. The strains belonged to three different PCR ribotypes: 001, 014/020/077 and 015. The detection thresholds for vegetative cells and spores cells varied between 1 and 3 c.f.u. in 20 g salad and between 6 and 15 c.f.u. in 20 g salad, respectively, for the method employed.

The attP region of the Clostridium difficile phage ϕCD27 was identified, located immediately downstream of the putative recombinase. The phage could integrate into two specific sites (attB) in the C. difficile genome, one of which was in an open reading frame encoding a putative ATPase of an ABC transporter and the other in an open reading frame encoding a putative ATPase of the flagella protein export apparatus. The prophage was capable of excision and formation of a circular molecule and phages were spontaneously released at a low frequency during growth. Infection and lysogeny of a C. difficile strain previously shown to be sensitive to ϕCD27 were demonstrated, leading to a reduction in toxin production. Finally, a putative repressor was identified which is likely to be involved in maintaining lysogeny in these strains.

An increased incidence of Clostridium difficile infection (CDI) is associated with the emergence of epidemic strains characterized by high genetic diversity. Among the factors that may have a role in CDI is a family of 29 paralogues, the cell-wall proteins (CWPs), which compose the outer layer of the bacterial cell and are likely to be involved in colonization. Previous studies have shown that 12 of the 29 cwp genes are clustered in the same region, named after slpA (cwp1), the slpA locus, whereas the remaining 17 paralogues are distributed throughout the genome. The variability of 14 of these 17 cwp paralogues was determined in 40 C. difficile clinical isolates belonging to six of the currently prevailing PCR ribotypes. Based on sequence conservation, these cwp genes were divided into two groups, one comprising nine cwp loci having highly conserved sequences in all isolates, and the other five loci showing low genetic conservation among isolates of the same PCR ribotype, as well as between different PCR ribotypes. Three conserved CWPs, Cwp16, Cwp18 and Cwp25, and two variable ones, Cwp26 and Cwp27, were characterized further by Western blot analysis of total cell extracts or surface-layer preparations of the C. difficile clinical isolates. Expression of genetically invariable CWPs was well conserved in all isolates, whilst genetically variable CWPs were not always expressed at comparable levels, even in strains containing identical sequences but belonging to different PCR ribotypes. This is the first report on the distribution and variability of a number of genes encoding CWPs in C. difficile.

Host anti-toxin immune responses play important roles in Clostridium difficile disease and outcome. The relationship between host immune and inflammatory responses during severe C. difficile infection (CDI) and the risk of mortality has yet to be defined. We aimed to investigate the host systemic IgG anti-toxin immune responses, the in vitro cytotoxicity of the infecting C. difficile ribotyped strain, and the host inflammatory markers and their relationship to CDI disease severity and risk of mortality. Inflammatory markers, co-morbidities and CDI outcomes were recorded in a prospective cohort of 150 CDI cases. Serum anti-cytotoxin A (TcdA) and anti-TcdB IgG titres were measured by ELISA and the infecting C. difficile isolate was ribotyped and the in vitro cytotoxin titre assessed. A low median anti-TcdA IgG titre was significantly associated with 30-day all-cause mortality (P<0.05). Ribotype 027 isolates were significantly more toxinogenic than other ribotypes (P<0.00001). High cytotoxin titres correlated with increased inflammatory markers but also higher anti-TcdA and -TcdB (P<0.05) IgG responses resulting in a lower risk of mortality. On multivariate analysis, predictors of mortality were peak white cell count >20×109 l−1 [odds ratio (OR) 11.53; 95 % confidence interval (CI) 2.38–55.92], creatinine concentration >133 µmol l−1 (OR 6.54; 95 % CI 1.47–29.07), Horn’s index >3 (OR 4.09; 95 % CI 0.76–22.18) and low anti-TcdA IgG (OR 0.97; 95 % CI 0.95–0.99), but not ribotype, cytotoxin titre or anti-TcdB IgG. Thus, host pro-inflammatory and humoral responses correlate with the cytotoxin titre of the infecting strain and effective anti-toxin immune responses reduce the risk of mortality.

In Clostridium difficile, resistance to the macrolide-lincosamide-streptogramin B group of antibiotics generally relies on erm(B) genes. In this study, we investigated elements with a genetic organization different from Tn5398, the mobilizable non-conjugative element identified in C. difficile strain 630. Our results suggested that the elements most frequently found in strains isolated during the European surveillance study in 2005 were related to Tn6194, the conjugative transposon recently detected in different C. difficile types, including PCR-ribotype 027. We characterized a Tn6194-like and a novel element rarely found in clinical isolates. A burden on the in vitro fitness of C. difficile was observed after the acquisition of these elements as well as of Tn5398.

Three hundred and thirty-five patients with laboratory-confirmed Clostridium difficile infections (CDIs) were studied for epidemiological features, clinical presentation and laboratory markers. They were followed up for 1 year to determine recurrence and mortality. Four hundred and thirty-two episodes were recorded. One year mortality was 41.8 % of which CDI was listed on 20 % of the death certificates. One year recurrence rate was 22.9 %. PCR ribotype 001 was the commonest epidemiological type and ribotype 027 was not detected. High total leucocyte count and low albumin were significantly associated with mortality, as was the absence of a GI-invasive procedure in the 12 weeks preceding CDI diagnosis, probably due to patients being unfit for the procedure. No association with acid suppressants, deletion in the tdcC anti-sigma factor or vancomycin-resistant enterococcus/methicillin-resistant Staphylococcus aureus co-infection was detected. One year mortality was higher in patients who developed recurrent infections (P<0.001). Differences in ribotype were observed in 2.3 %, 11.11 %, 20 % and 32.4 % isolates with time intervals between sampling of 0–20, 21–40, 41–60 and >60 days, respectively, suggesting that the arbitrary cut-off of 28 days to call a repeat infection a reinfection may not be correct in some cases.

A total of 188 human (n = 92) and animal (n = 96) isolates of Clostridium difficile of different PCR ribotypes were screened for susceptibility to 30 antimicrobials using broth microdilution. When comparing the prevalence of antimicrobial resistance, the isolates of animal origin were significantly more often resistant to oxacillin, gentamicin and trimethoprim/sulfamethoxazole (P<0.01). The most significant difference between the animal and human populations (P = 0.0006) was found in the level of imipenem resistance, with a prevalence of 53.3 % in isolates of human origin and 28.1 % in isolates of animal origin. Overall, the results show similar MICs for the majority of tested antimicrobials for isolates from human and animal sources, which were collected from the same geographical region and in the same time interval. This supports the hypothesis that C. difficile could be transmissible between human and animal hosts. Resistant isolates have been found in all animal species tested, including food and companion animals, and also among non-toxigenic isolates. The isolates of the most prevalent PCR ribotype 014/020 had low resistance rates for moxifloxacin, erythromycin, rifampicin and daptomycin, but a high resistance rate for imipenem. Multiresistant strains were found in animals and humans, belonging to PCR ribotypes 012, 017, 027, 045, 046, 078 and 150, and also to non-toxigenic strains of PCR ribotypes 010 and SLO 080.

Two cases of fulminant colitis due to Clostridium difficile occurred within ten weeks of each other on the same ward of a hospital in Japan. The patients died 2 and 4 days after the onset of colitis. C. difficile isolates obtained from both patients were toxin A-positive, toxin B-positive and binary toxin-positive. These isolates yielded identical results by both PCR ribotyping and slpA sequence typing. However, the banding patterns and slpA sequences of the isolates differed from those of PCR ribotype 027, as well as those of PCR ribotype 078. The tcdC sequences of the isolate differed from those of C. difficile 027, but a single base-pair deletion at position 117 and an 18 bp deletion, both of which were identical to the sequence of the reference strain of 027, were found. This type may be a new hypervirulent strain, but further studies of the epidemiology and pathogenicity of the strain are needed.