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PubMed1999

Detection and transcription of toxin DNA in a nontoxigenic strain of Clostridium difficile.

Genomic DNA from three Clostridium difficile strains was analyzed by PCR for DNA sequences encoding toxin A (tcdA) and toxin B (tcdB). Toxigenic control strain VPI 10463 possessed tcdA, tcdB, and an open reading frame (tcdE) between these two genes, whereas nontoxigenic control strain 85 lacked each of these genetic determinants. However, strain M90, also a nontoxigenic strain, was found to possess tcdA, tcdB, and tcdE. Normally the presence of toxin genes is associated with toxigenicity. Analysis of tcdA and tcdB mRNA revealed toxin gene transcription in strains VPI 10463, 23 (a mildly toxigenic strain), and M90, but not in strain 85. However, for strain M90, tcdA and tcdB mRNA was at the lower limit of detection, whereas mRNAs encoding tcdA and tcdB were easily detected in strains VPI 10463 and 23. Low levels of toxin gene transcription is the probable cause of M90's lack of toxigenicity.

Bacterial Proteins↗
PubMed1995

Glucosylation of Rho proteins by Clostridium difficile toxin B.

Toxin A and B, the major virulence factors of Clostridium difficile, are the causative agents of antibiotic-associated pseudomembranous colitis. In cultured cell lines their potent cytotoxicity results from their ability to induce disaggregation of the microfilament cytoskeleton. Toxin B acts on the low-molecular-mass GTPase RhoA, which is involved in the regulation of the actin cytoskeleton. We report here that toxin B catalyses the incorporation of up to one mole of glucose per mole of RhoA at the amino acid threonine at position 37. The modification was identified and localized by tandem electrospray mass spectrometry. UDP-glucose selectively serves as cosubstrate for the monoglucosylation reaction catalysed by toxin B. Microinjection of RhoA previously glucosylated by toxin B into monolayer cells caused disaggregation of actin filaments, indicating a dominant-negative activity of glucosylated RhoA.

Actins↗
PubMed2001

Intestinal epithelial damage in sids babies and its similarity to that caused by bacterial toxins in the rabbit.

Sections of the duodenum, jejunum, ileum, caecum and large intestine from 14 sudden infant death syndrome (SIDS) babies were examined by scanning (SEM) and transmission electron microscopy (TEM). The type and amount of damage was characterised and quantitated and compared with the presence of Clostridium perfringens, Clostridium difficile, Escherichia coli and Staphylococcus aureus in faecal samples from the babies and toxins from the bacteria in faecal samples and serum from the babies. The data were compared with the damage that these toxins cause to the rabbit intestinal epithelium (see the previous paper in this issue). Damage was present in most of the SIDS samples, varying from 0 to 96%, and most damage occurred when the faecal samples contained the above bacteria and their toxins. Damage varied from removal of microvilli, damage to villus tips, separation of and removal of epithelial cells from the lamina propria, and removal of enterocytes leaving goblet and tuft cells, to damage and breakdown of the lamina propria. The results support the hypothesis that the cause of death in a significant proportion of SIDS babies may result from the absorption of toxins from the intestinal tract initiating a toxic shock reaction.

Animals↗
PubMed1992

Comparative study of immunological properties and cytotoxic effects of Clostridium difficile toxin B and Clostridium sordellii toxin L.

We compared the immunological properties and cytotoxic effects of Clostridium difficile toxin B and Clostridium sordellii toxin L. These two cytotoxins are immunologically related in that the cytotoxic effect of either toxin can be neutralized by the polyclonal antiserum prepared against either cytotoxin. On the other hand, polyclonal antiserum prepared against Clostridium difficile enterotoxin A did not cross-react with the cytotoxins B and L when tested by cytotoxic neutralization test nor by double immunodiffusion assay. However, despite this immunological relationship between toxins B and L, the morphological modifications observed in MacCoy cells induced by treatment with these cytotoxins are clearly distinct. We describe the first quantitative analysis of specific cellular parameters which illustrates the morphological differences induced by these cytotoxins. Moreover, immunocytochemical experiments show that, whereas disruption of microfilaments is observed with toxin B- and L-treatments, alterations of F-actin network are different in the cells treated with toxin B or L. The observation that the cellular modifications induced by toxin B- and toxin L-treatment differ suggests that the molecular mechanisms involved in the respective cytotoxicities are also likely to be different.

Actin Cytoskeleton↗
PubMed2006

Prevalence and clinical features of Clostridium difficile-associated diarrhea in a tertiary hospital in northern Taiwan.

BACKGROUND AND PURPOSE: Although the clinical manifestations of and risk factors for Clostridium difficile-associated diarrhea (CDAD) have been extensively investigated in western populations, data from Taiwanese patients are comparatively limited. This study investigated the incidence density of CDAD in Taiwanese patients and also the risk factors and clinical manifestations of CDAD. METHODS: From September 21, 2003 to December 21, 2003, patients hospitalized in 2 infection wards and 6 medical intensive care units at National Taiwan University Hospital who were older than 20 years, had a history of antibiotic usage within the prior 6 weeks, and developed diarrhea without another identified etiology were classified as having antibiotic-associated diarrhea (AAD), and were enrolled for further study. The diagnosis of CDAD was established when toxin A of C. difficile was detected in stool. RESULTS: The incidence density of AAD was 1/100 person-days of antibiotics usage. CDAD accounted for 12.5% of AAD. Fever and abdominal discomfort developed in only less than half of CDAD patients. Pus cell in the stool sample was found in 100 percent of patients with CDAD. Univariate analysis revealed that presence of malignancy and treatment with antifungal agents within the previous 6 weeks were risk factors for CDAD development. In multivariate analysis, use of antifungal agents was the only independent risk factor for CDAD. CONCLUSION: The incidence density of CDAD in this study of Taiwanese patients with AAD was 12.5%. Prior usage of antifungal agents was the only independent factor associated with subsequent CDAD development in patients with AAD.

Adult↗
PubMed2003

Six rapid tests for direct detection of Clostridium difficile and its toxins in fecal samples compared with the fibroblast cytotoxicity assay.

Clostridium difficile is one of the most frequent causes of nosocomial gastrointestinal disease. Risk factors include prior antibiotic therapy, bowel surgery, and the immunocompromised state. Direct fecal analysis for C. difficile toxin B by tissue culture cytotoxin B assay (CBA), while only 60 to 85% sensitive overall, is a common laboratory method. We have used 1,003 consecutive, nonduplicate fecal samples to compare six commercially available immunoassays (IA) for C. difficile detection with CBA: Prima System Clostridium difficile Tox A and VIDAS Clostridium difficile Tox A II, which detect C. difficile toxin A; Premier Cytoclone A/B and Techlab Clostridium difficile Tox A/B, which detect toxins A and B; and ImmunoCard Clostridium difficile and Triage Micro C. difficile panels, which detect toxin A and a species-specific antigen. For all tests, Triage antigen was most sensitive (89.1%; negative predictive value [NPV] = 98.7%) while ImmunoCard was most specific (99.7%; positive predictive value [PPV] = 95.0%). For toxin tests only, Prima System had the highest sensitivity (82.2%; NPV = 98.0%) while ImmunoCard had the highest specificity (99.7%; PPV = 95.0%). Hematopoietic stem cell transplant (HSCT) patients contributed 44.7% of all samples tested, and no significant differences in sensitivity or specificity were noted between HSCT and non-HSCT patients. IAs, while not as sensitive as direct fecal CBA, produce reasonable predictive values, especially when both antigen and toxin are detected. They also offer significant advantages over CBA in terms of turnaround time and ease of use.

Bacterial Toxins↗
PubMed2003

[Fungemia after oral treatment with Saccharomyces boulardii in a patient with multiple comorbidities].

HISTORY AND CLINICAL FINDINGS: A 48-year-old diabetic with multiple co-morbidities presented with generalized micro- and macroangiopathy including peripheral artery disease stage IV with necroses in several digits of both feet. He was admitted to the department of surgery for the insertion of femoropopliteal bypasses. INVESTIGATIONS: Infectious parameters were elevated (CRP 66.1 mg/l, sedimentation rate 90/96), accompanied by anemia (Hb 7.1 mmol/l), leukocytosis (14.8 Gpt/l) and thrombocytosis (514 Gpt/l). Body temperature was normal (36.8 degrees C). With insulin treatment the patient became nearly normoglycemic (HbA1c 6.8 %). TREATMENT AND FOLLOW UP: After receiving different broad-spectrum antibiotics over seven weeks the patient developed Clostridium difficile toxin-positive diarrhea that resolved after administration of oral metronidazole and Saccharomyces boulardii (Perenterol ((R))). Three days after bypass insertion, both legs had to be amputated due to infection and beginning sepsis. The condition of the patient improved. However, eight days after bypass-insertion the patient developed a toxic megacolon and sepsis. Blood cultures yielded the growth of Saccharomyces cerevisae. Despite of intensive care treatment the patient died five days later from to multi-organ failure. CONCLUSION: S. boulardii (synonym: S. cerevisiae) is considered an non-pathogenic probiotic yeast, and live yeast cells are used for supportive therapy of diarrhea. The present case and a review of the literature demonstrate that fungemia and sepsis are rare complications of the administration of S. boulardii in immunocompromised patients. For this reason the therapeutic usage of probiotics should be carefully considered regarding its risk-benefit potential.

Administration, Oral↗
PubMed1998

CGRP upregulation in dorsal root ganglia and ileal mucosa during Clostridium difficile toxin A-induced enteritis.

We have previously reported that pretreatment of rats with capsaicin (an agent that ablates sensory neurons) or CP-96345 (a substance P receptor antagonist) dramatically inhibits fluid secretion and intestinal inflammation in ileal loops exposed to Clostridium difficile toxin A. The aim of this study was to determine whether calcitonin gene-related peptide (CGRP), a neuropeptide also found in sensory afferent neurons, participates in the enterotoxic effects of toxin A. Administration of toxin A was also found to increase CGRP content in dorsal root ganglia and ileal mucosa 60 min after toxin exposure. Furthermore, immunohistochemical studies demonstrated increased neuronal staining for CGRP 2 h after toxin A treatment. Pretreatment of rats with CGRP-(8-37), a specific CGRP antagonist, before instillation of toxin A into ileal loops significantly inhibited toxin-mediated fluid secretion (by 48%), mannitol permeability (by 83%), and histological damage. We conclude that CGRP, like substance P, contributes to the secretory and inflammatory effects of toxin A via increased production of this peptide from intestinal nerves, including primary sensory afferent neurons.

Animals↗
PubMed2006

Risk factors for Clostridium difficile-associated diarrhea on an adult hematology-oncology ward.

Nosocomial diarrhea caused by Clostridium difficile causes significant morbidity and mortality in an increasing proportion of hospitalized patients annually. This case-control study of patients admitted to the hematology-oncology ward of a tertiary academic medical center over a 2-year period demonstrates that patients with Clostridium difficile-associated diarrhea (CDAD) were 22 times more likely than ward-matched controls with diarrhea to have received any antibiotic either during hospitalization or in the month preceding admission (p < 0.005), and they were nearly three times as likely as controls to have received a cephalosporin during the same period (p < 0.005). Diarrhea among lung cancer patients was approximately three times more likely to be caused by this organism than to be due to other causes (p = 0.04). A trend towards CDAD patients receiving higher numbers of different antibiotics during hospitalization (3.3 vs. 2.6, 95%CI -1.42-0.02, p = 0.06) was noted. Administration of interleukin-2 either during hospitalization or in the 30 days preceding admission was seven times more likely to have occurred in CDAD cases (p = 0.04), raising the question of whether or not this agent increases risk.

Adult↗
PubMed2001

Evidence for holin function of tcdE gene in the pathogenicity of Clostridium difficile.

Toxigenic strains of Clostridium difficile produce two large bacterial toxins called toxins A (TcdA) and B (TcdB). tcdA and tcdB genes are located on the pathogenicity locus of C. difficile, a unique characteristic of toxigenic strains of this species. Intergenic to the two toxin genes is tcdE, a small 501-bp open reading frame of unknown function. Expression of the tcdE gene in Escherichia coli caused bacterial cell death. Computational analysis of the amino acid sequence of TcdE revealed structural features that are strikingly similar to a class of bacteriophage proteins called holins. Holins are cytolytic proteins that cause lysis of bacterial hosts to effect the release of progeny phages. Further analysis of the recombinant clone expressing TcdE by transmission electron microscopy confirmed that the site of action of TcdE is on the bacterial cell membrane. The results provide evidence that TcdE is structurally and functionally similar to holin proteins. TcdE may function as a lytic protein to facilitate the release of TcdA and TcdB to the extracellular environment, as these toxins lack signal peptide.

Amino Acid Sequence↗
PubMed2001

Variation in the surface layer proteins of Clostridium difficile.

Surface layers (S-layers) form regular crystalline structures on the outermost surface of many bacteria. Clostridium difficile possesses such an S-layer consisting of two protein subunits. Treatment of whole cells of C. difficile with 5 M guanidine hydrochloride revealed two major proteins of different molecular masses characteristic of the S-layer on SDS-PAGE. In this study 25 isolates were investigated. A high degree of variability in the molecular mass of the two S-layer proteins was evident. Molecular masses ranged from 48 to 56 kDa for the heavier protein and from 37 to 45 kDa for the lighter protein. A further protein component of 70 kDa was detectable in all isolates. No cross-reaction was seen between the two major proteins from isolates that produced different S-layer patterns, and most S-layer proteins from isolates with the same or similar banding patterns did not cross-react. The S-layer proteins, when detected by a combination of Coomassie blue staining and immunoblotting, are a useful marker for phenotyping.

Animals↗
PubMed1996

[Current diagnosis, clinical course and treatment of acute colitis infection with Clostridium difficile].

Detection rate of G. intestionalis in feces with direct microscopy has been compared with the immuno-enzymatic technique detecting protein GSA 65 with Alexon Inc., ProSpec T/Giardia reagents kit. The results obtained with both methods have further been compared with those obtained by microscopic examination of the duodenal content. Detectability of Giardia intestinalis with EIA technique with the use of ready-made kit has been assessed. Feces have been collected from 371 patients. Protein GSA 65 has been present in 170 samples, 45.8%, examined with the use of ProSpec T/Gardia kit. Giardia intestinalis cysts have been detected microscopically in the feces of 37 patients, i.e. in 22.3%. Microscopic examinations carried out by three independent examiners have shown marked diversity in the rate of positive results, being 0.1% (examiner A), 28.6% (B), and 45.2% (C). Comparison of G. intestinale detectability of all 3 techniques used have shown absence of protein GSA 65 in 2 out of 9 examined patients. However, trophozoits have been present in the duodenal content. Test performed with kits made by Alexon Inc. and DIALAB have given 45.8% and 60.7% of the positive results, respectively.

Adult↗
PubMed1998

[Systematic detection of toxigenic strains of Clostridium difficile--is it useful?].

The incidence of Clostridium difficile (Cd) infection is rising and Cd in fact is now endemic in many hospitals. During the past 4 years we analyzed our data concerning diarrhea caused by Cd in our 700 beds hospital. A positive case was defined as a Cd cytotoxine positive with or without positive culture for Cd. In the present study 120 episodes of Cd associated diarrhea occurred in 102 patients. 1101 stools were cultured from 921 patients. Since 1995 we choose to systematically evaluate Cd in diarrheal stools from hospitalized patients. 120 stool were positives (102 patients), we observed a significant difference between the 2 study periods: Cd was recovered from 16.9% of stool specimen during 1993-1994 and from 9.6% since 1995. This study clearly confirm the common role of Cd in our hospitalized patients as in all positive case, Cd was the only enteropathogen isolated. We suggest the systematic investigation of Cd in hospitalized patients.

Bacterial Proteins↗
PubMed1999

[Susceptibility of Clostridium difficile to metronidazole using the E-test: effect of the culture medium].

The treatment of intestinal Clostridium difficile infections rests on administration of either a glycopeptide or metronidazole. Given the current shifts in resistance patterns of anaerobes to antimicrobials, a study of the susceptibility of C. difficile to metronidazole was timely. The objective of this study was to evaluate the influence of the culture medium on the Minimal Inhibitory Concentration (MIC) of metronidazole as determined using the E-test. Thirty-one strains were grown on three different media supplemented with 5% horse blood, namely Columbia agar, Wilkens Chalgren agar, and Brucella agar. Results were compared to those obtained using the reference agar dilution method (ADM). As recommended by the French Society for Microbiology, susceptibility was defined as an MIC < or = 4 mg/L. When used on strains susceptible by the ADM, the E-test yielded lower values than the ADM with all three media. Furthermore, findings suggest that E-test results obtained with strains whose MIC is in the 4 to 8 mg/L range by the ADM should be interpreted with caution and, in some cases, tested using the ADM.

Animals↗
PubMed1994

Clostridium difficile toxin A therapy for HCT 116 human colon cancer in nude mice.

Clostridium difficile toxin A was evaluated for an antitumor effect in vivo on HCT 116 human colon carcinoma cells growing subcutaneously in nude mice. A mean reduction in tumor volume of at least 65%, by measurement in three dimensions, was observed in mice who received two 9- to 13-day courses of daily intraperitoneal injections of toxin A as compared to mice receiving diluent alone. Reversible adverse effects of toxin A were noted in some animals, consisting primarily of liver toxicity and skin rash. HCT 116 cells in toxin A-treated mice grew as flattened tumors with ulcerated centers compared to rounded tumors without ulceration in controls. Histologic examination of tumors from representative mice revealed that two thirds of the tumor in a treated mouse was necrotic compared to only one third in a control, suggesting greater antitumor efficacy of toxin A than estimated by tumor measurements alone.

Animals↗
PubMed1992

Characterization of a toxin A-negative, toxin B-positive strain of Clostridium difficile.

This study was undertaken to examine toxin production by Clostridium difficile 8864, a naturally occurring isolate that has been reported to produce toxin B in the absence of toxin A. To date, this is the only strain of C. difficile reported to produce only one of the toxins. The results of our initial studies with antibodies against toxins A and B confirmed these observations. Toxin B from strain 8864 and from VPI strain 10463, a strain that produces high levels of both toxin A and toxin B, was purified to homogeneity by sequential anion-exchange chromatography on DEAE-Sepharose CL-6B, gel filtration on Ultrogel AcA22, and immunoadsorption chromatography, and their toxic activities were compared. Our results showed that toxin B from strain 8864 and toxin B from C. difficile VPI strain 10463 were comparable in their cytotoxic activities and that the 8864 toxin B was more lethal. In addition, we observed that toxin B from strain 8864 was weakly enterotoxic, which may explain the ability of this strain to cause intestinal disease in hamsters treated with antibiotics. Analysis with specific antibodies showed that the toxin B molecules from these strains were highly related but contained distinct epitopes. The results of hybridization studies with probes specific for the toxin B gene of VPI strain 10463 demonstrated differences between the toxin B genes of the two strains. In addition, probes specific for the toxin A gene of VPI strain 10463 showed that strain 8864 contains a region which shows identity with the 5' end of the toxin A gene but not the region of the gene which encodes a hydrophobic region and the repeating units.

Bacterial Proteins↗
PubMed2002

Morbidity, mortality, and healthcare burden of nosocomial Clostridium difficile-associated diarrhea in Canadian hospitals.

OBJECTIVE: To assess the healthcare burden, morbidity, and mortality of nosocomial Clostridium difficile-associated diarrhea (N-CDAD) in Canadian hospitals. DESIGN: Laboratory-based prevalence study. SETTING: Nineteen acute-care Canadian hospitals belonging to the Canadian Hospital Epidemiology Committee surveillance program. PATIENTS: Hospitalized patients in the participating centers. METHODS: Laboratory-based surveillance was conducted for C. difficile toxin in stool among 19 Canadian hospitals from January to April 1997, for 6 continuous weeks or until 200 consecutive diarrhea stool samples had been tested at each site. Patients with N-CDAD had to fulfill the case definition. Data collected for each case included patient demographics, length of stay, extent of diarrhea, complications of CDAD, CDAD-related medical interventions, patient outcome, and details of death. RESULTS: We found that 371 (18%) of 2,062 tested patients had stools with positive results for C difficile toxin, of whom 269 (13%) met the case definition for nosocomial CDAD. Of these, 250 patients (93%) had CDAD during their hospitalization, and 19 (7%) were readmitted because of CDAD (average readmission stay, 13.6 days). Forty-one patients (15.2%) died, of whom 4 (1.5% of the total) were considered to have died directly or indirectly of N-CDAD. The following N-CDAD-related morbidity was noted: dehydration, 3%; hypokalemia, 2%; gastrointestinal hemorrhage requiring transfusion, 1%; bowel perforation, 0.4%; and secondary sepsis, 0.4%. The cost of N-CDAD readmissions alone was estimated to be a minimum of $128,200 (Canadian dollars) per year per facility. CONCLUSION: N-CDAD is a common and serious nosocomial infectious complication in Canada, is associated with substantial morbidity and mortality, and imposes an important financial burden on healthcare institutions.

Aged↗
PubMed1992

A non-haemagglutinating form of Clostridium difficile toxin A.

Analysis of crude culture filtrate of Clostridium difficile by Mono Q-anion exchange fast protein liquid chromatography (FPLC) demonstrated that toxin A had distinct peaks of activity for cytotoxicity and haemagglutination, as also did highly purified toxin A obtained by thyroglobulin affinity chromatography (TG) followed by two sequential anion-exchange chromatographic steps with Q-Sepharose FF and Mono Q. From TG unbound fractions a highly cytotoxic but weakly haemagglutinating variant (toxin A') of toxin A was obtained by Q-Sepharose FF and Mono Q chromatography. Analysis of toxins A and A' from cultures of C. difficile in a chemically defined medium, and of toxin A dialysed against brain heart infusion broth, indicated that A' was not merely toxin A coupled to a component of the growth medium. Polyacrylamide gel electrophoresis under non-denaturing conditions showed that toxins A and A' had the same Mr. Immunoblotting with mouse monospecific A antitoxin showed that five bands larger than the major 240-Kda band were more strongly developed in toxin A than in A' in denaturing but non-reducing conditions, and in reducing conditions eight bands (38-175 Kda) were seen in toxin A but not A'. Immunoblotting with a monoclonal antibody (PCG-4) showed that, in both reducing and non-reducing conditions, two bands of 160 and 155 Kda were more prominent in toxins A and A' respectively, and four bands (195, 180, 175 and 125 Kda) were detected only in toxin A'.

Animals↗