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PubMed2003

Molecular analysis of Clostridium difficile strains isolated from 18 cases of recurrent clostridium difficile-associated diarrhea.

Recurrence of Clostridium difficile-associated diarrhea (CDAD) occurs in 15 to 20% of patients after discontinuation of treatment. Arbitrarily primed PCR was used to investigate the epidemiology of recurrent CDAD in 18 patients. Reinfection with a new strain occurred in 6 of 18 patients (33.3%), while 12 patients relapsed with the original strain shortly after discontinuation of treatment. These data suggest that reinfection with exogenous C. difficile is a common problem and that not all recurrences are due to relapse.

Bacterial Typing Techniques↗
PubMed2004

Multiplex PCR targeting tpi (triose phosphate isomerase), tcdA (Toxin A), and tcdB (Toxin B) genes for toxigenic culture of Clostridium difficile.

A multiplex PCR toxigenic culture approach was designed for simultaneous identification and toxigenic type characterization of Clostridium difficile isolates. Three pairs of primers were designed for the amplification of (i) a species-specific internal fragment of the tpi (triose phosphate isomerase) gene, (ii) an internal fragment of the tcdB (toxin B) gene, and (iii) an internal fragment of the tcdA (toxin A) gene allowing distinction between toxin A-positive, toxin B-positive (A+B+) strains and toxin A-negative, toxin B-positive (A-B+) variant strains. The reliability of the multiplex PCR was established by using a panel of 72 C. difficile strains including A+B+, A-B-, and A-B+ toxigenic types and 11 other Clostridium species type strains. The multiplex PCR assay was then included in a toxigenic culture approach for the detection, identification, and toxigenic type characterization of C. difficile in 1,343 consecutive human and animal stool samples. Overall, 111 (15.4%) of 721 human samples were positive for C. difficile; 67 (60.4%) of these samples contained A+B+ toxigenic isolates, and none of them contained A-B+ variant strains. Fifty (8%) of 622 animal samples contained C. difficile strains, which were toxigenic in 27 (54%) cases, including 1 A-B+ variant isolate. Eighty of the 721 human stool samples (37 positive and 43 negative for C. difficile culture) were comparatively tested by Premier Toxins A&B (Meridian Bioscience) and Triage C. difficile Panel (Biosite) immunoassays, the results of which were found concordant with toxigenic culture for 82.5 and 92.5% of the samples, respectively. The multiplex PCR toxigenic culture scheme described here allows combined diagnosis and toxigenic type characterization for human and animal C. difficile intestinal infections.

Animals↗
PubMed2006

Correlation of disease severity with fecal toxin levels in patients with Clostridium difficile-associated diarrhea and distribution of PCR ribotypes and toxin yields in vitro of corresponding isolates.

We investigated in vivo and in vitro yields of toxins A and B from and PCR ribotypes of Clostridium difficile isolates from 164 patients with differing severities of C. difficile-associated diarrhea (CDAD) (patients were grouped as follows: <3 loose stools per day, n = 45; 3 to 10 per day, n = 97; >10 per day, n = 22). The median fecal toxin levels in each group were 0.5, 6.8, and 149 U/g feces (P < 0.001), respectively. Patients with severe diarrhea also had more-frequent occurrence of blood in stool and vomiting, but there was no association with fecal toxin levels per se. There was no correlation between fecal toxin level and toxin yield in vitro for the corresponding C. difficile isolate or between its PCR ribotype and disease severity. A broad range of toxin yields among isolates belonging to major PCR ribotypes indicated a presence of many subtypes. We hypothesize that bacterial and host factors that affect C. difficile toxin levels in feces are important determinants of symptoms in CDAD patients. An inverse correlation between toxin yield and spore count (r = 0.66) in stationary-phase cultures supported the notion that toxin production and sporulation represent opposite alternative survival strategies for C. difficile cells facing nutrient shortage.

Adolescent↗
PubMed1996

Nosocomial empyema caused by Clostridium difficile.

Pleural infection with Clostridium difficile is extremely rare. A case of nosocomial empyema following chest drain insertion in a 46 year old man is described. The potential of C difficile to cause extra-intestinal infections should be recognised and its isolation from other sites should not be ignored.

Clostridioides difficile↗
PubMed1997

IL-8 release and neutrophil activation by Clostridium difficile toxin-exposed human monocytes.

Neutrophil infiltration is central to the pathogenesis of Clostridium difficile toxin A-induced enterocolitis. This study examines whether monocyte activation by C. difficile toxins is instrumental in initiating neutrophil activation and recruitment. Human monocytes were exposed to low concentrations of highly purified C. difficile toxins, and the conditioned media were harvested for cytokine and functional assays. Monocytes exposed to C. difficile toxin A (10(-10) M) or toxin B (10(-12) M) released 100 and 20 times basal levels, respectively, of the neutrophil chemoattractant interleukin-8 (IL-8). Reverse transcriptase-polymerase chain reaction demonstrated a marked increase in IL-8 mRNA expression by monocytes 3 h after toxin exposure. Conditioned media from toxin A- and toxin B-treated monocytes stimulated neutrophil migration (324 and 245% of control, respectively). This effect was completely blocked by IL-8 antiserum. These media also upregulated neutrophil CD11b/CD18 and endothelial cell intercellular adhesion molecule-1 expression. C. difficile toxins, at low concentrations, potently activate monocytes to release factors, including IL-8, that facilitate neutrophil extravasation and tissue infiltration. Our findings indicate a major role for toxin-mediated monocyte and macrophage activation in C. difficile colitis.

Bacterial Proteins↗
PubMed2004

Clostridium difficile: a questionnaire survey of laboratory practice in England, Wales, and Northern Ireland.

Since January 2004, the incidence of Clostridium difficile associated disease (CDAD) has been monitored by a systematic, national, laboratory surveillance system. This system incorporates the recommendations of a body of experts, the National Clostridium difficile Standards Group, which was convened in 2002 to advise the Department of Health (DoH). The recommendations of the group were informed by a questionnaire survey of current practice, and the results of that survey have been used to assess the implications of the recommendations on laboratory practice. Large variability was found as to the specimens selected, tested, and reported on for C. difficile. Standardisation of the diagnosis and reporting of CDAD is desirable and necessary to increase understanding of its epidemiology.

Clinical Laboratory Techniques↗
PubMed2006

[A case of multiple recurrence of Clostridium difficile-associated diarrhea--analysis of isolates from the patient using PCR ribotyping].

Recurrence of Clostridium difficile-associated diarrhea (CDAD) is a serious and still unsolved problem. Little is known about the precise mechanism of the recurrence with CDAD. To elucidate the issue, we analyzed C. difficile strains obtained from the patient with multiple recurrence of CDAD. A 72-year-old female received rectoidectomy was developed CDAD after administration of cefmetazole sodium for 5 days. She was recovered from illness by the administration 2g of vancomycin hydrochloride for 2 weeks. However she was recurred CDAD twice within 2 months thereafter. Six C. difficile isolates recovered from the stool specimens obtained during each episode were typed by polymerase chain reaction amplifying of rRNA intergenic spacer regions 8 PCR ribotyping was carried out. The PCR ribotype of the first episode was identical (ribotype smz) to that of the second episode, and the PCR ribotype of the third were distinct (ribotype hr) from those on previous two episodes. These findings indicate that the second episode was caused by the reactivation of continuously infected C. difficile strain (relapse) and the third episode was by reinfection of the new

Aged↗
PubMed1990

Cloning of Clostridium difficile toxin B gene and demonstration of high N-terminal homology between toxin A and B.

High titered Clostridium sordellii lethal toxin antiserum, cross-reactive with C. difficile cytotoxin B (ToxB), was used to isolate toxB fragments from a C. difficile expression library. Recombinant clones containing toxB fragments of the 5' and 3' end were isolate. A 2.5-kb HincII fragment of chromosomal DNA overlaps both groups of clones. A partial restriction map of the total toxB gene is presented. The gene is positioned upstream of utxA and toxA, toxB has a size of 6.9 kb, corresponding to a 250-kDa polypeptide. A partial sequence of the 5' end of toxB was determined. The sequence contains 398 bp upstream of toxB with a putative Shine-Dalgarno box (AGGAGA) and 609 bp of the toxB open reading frame. The N-terminal 203 amino acids of ToxB were compared with the N-terminal amino acids of the enterotoxin A (ToxA). A homology of 64% of the residues was detected, which proves the relatedness of ToxA and ToxB of C. difficile.

Amino Acid Sequence↗
PubMed1992

Evaluation of a new commercial Clostridium difficile toxin A enzyme immunoassay using diarrhoeal stools.

A new, commercially available enzyme immunoassay for the detection of toxin A in stool specimens, the Premier Clostridium difficile Toxin A test (Meridian Diagnostics), was evaluated using 228 diarrhoeal stool specimens. Using a cytotoxin assay on HeLa cells as the reference method, this new test resulted in a sensitivity of 88% and a specificity of 95%. Using the presence or absence of a toxigenic strain in the stools as the reference method, the sensitivity was similar to that of the cytotoxin assay (71.7+ versus 70.5%) and the overall correlation was even better (89.4% versus 82%). The Premier Clostridium difficile Toxin A assay is rapid and easy to perform and is an excellent alternative to the usual toxin B assay.

Bacterial Toxins↗
PubMed2007

Evaluation of three rapid assays for detection of Clostridium difficile toxin A and toxin B in stool specimens.

Diagnosis of Clostridium difficile-associated disease continues to be difficult for clinical microbiology laboratories. The aim of this study was to evaluate the performance of three enzyme immunoassays for detection of C. difficile toxins A and B: the recently marketed rapid enzyme immunoassay Ridascreen Clostridium difficile Toxin A/B (R-Biopharm, Darmstadt, Germany) and two established enzyme immunoassays, the C. difficile Tox A/B II Assay (TechLab, Blacksburg, VA, USA) and the ProSpecT C. difficile Toxin A/B Microplate Assay (Remel, Lenexa, KS, USA). Stool specimens (n = 383) from patients with a clinical diagnosis of antibiotic-associated diarrhea were examined by these three enzyme immunoassays and were additionally cultured for C. difficile on selective agar. Samples giving discordant enzyme immunoassay results underwent confirmatory testing by tissue culture cytotoxin B assay and by PCR for toxin A (tcdA) and toxin B (tcdB) genes from C. difficile. Using the criteria adopted for this study, 60 (15.7%) samples tested positive for toxins A and/or B. Sensitivity and specificity of the enzyme immunoassays were, respectively, 88.3 and 100% for the TechLab enzyme immunoassay, 91.7 and 100% for the R-Biopharm enzyme immunoassay, and 93.3 and 100% for the Remel enzyme immunoassay. The differences between these results are statistically not significant (p > 0.05). The results show that all three enzyme immunoassays are acceptable tests for the detection of C. difficile toxins A and B directly in fecal specimens or in toxigenic cultures.

Bacterial Proteins↗
PubMed1996

A group II intron in a conjugative transposon from the gram-positive bacterium, Clostridium difficile.

We have been studying the conjugative transposon Tn5397, originally isolated from the Gram-positive pathogen Clostridium difficile. Physical analysis of this transposon demonstrated that it contained a group II intron. This is the first report of an intron in a conjugative transposon and the first report of a group II intron in Gram-positive bacteria. The intron interrupted a gene in Tn5397 that is almost identical to orf14 from Tn916. DNA hybridisation analysis showed that elements related to Tn5397, containing the group II intron, were present in five other C. difficile strains from different geographical locations suggesting that the element is likely to be widely distributed.

Cloning, Molecular↗
PubMed1998

Timing of surgery for fulminating pseudomembranous colitis.

BACKGROUND: With increasing antibiotic usage Clostridium difficile colitis is becoming more common. Surgery for fulminating C. difficile colitis, however, is rare because of the effectiveness of specific anticlostridial chemotherapy. Surgical outcome in five patients with fulminating C. difficile colitis involved in a recent outbreak of this disease is reported. METHODS: Five of 138 patients developed fulminating C. difficile colitis unresponsive to medical therapy. All patients had antibiotics in the preceding period. Indications for operation in those who underwent surgery were systemic toxicity with a pyrexia, marked leukocytosis and abdominal signs leading to progressive organ failure, despite appropriate anticlostridial antibiotic therapy. RESULTS: At operation all patients had a markedly oedematous colon with normal serosa but with acute mucosal colitis. All underwent subtotal abdominal colectomy and ileostomy. Progressive organ failure persisted in four, leading to death, giving a mortality rate of four in five in the operated group in comparison with 3.8 per cent (five of 133 patients) in those treated medically. CONCLUSIONS: These results indicate that this increasingly common disease frequently leads to a fatal outcome in patients requiring surgery and implies that earlier surgical consultation may be necessary to improve survival in patients with fulminating C. difficile colitis unresponsive to antibiotic therapy.

Abdomen, Acute↗
PubMed1995

The enterotoxin from Clostridium difficile (ToxA) monoglucosylates the Rho proteins.

The enterotoxin from Clostridium difficile (ToxA) is one of the causative agents of the antibiotic-associated pseudomembranous colitis. In cultured monolayer cells ToxA exhibits cytotoxic activity to induce disassembly of the actin cytoskeleton, which is accompanied by morphological changes. ToxA-induced depolymerization of actin filaments is correlated with a decrease in the ADP-ribosylation of the low molecular mass GTP-binding Rho proteins (Just, I., Selzer, J., von Eichel-Streiber, C., and Aktories, K. (1995) J. Clin. Invest. 95, 1026-1031). Here we report on the identification of the ToxA-induced modification of Rho. Applying electrospray mass spectrometry, the mass of the modification was determined as 162 Da, which is consistent with the incorporation of a hexose into Rho. From several hexoses tested UDP-glucose selectively served as cosubstrate for ToxA-catalyzed modification. The acceptor amino acid of glucosylation was identified from a Lys-C-generated peptide by tandem mass spectrometry as Thr-37. Mutation of Thr-37 to Ala completely abolished glucosylation. The members of the Rho family (RhoA, Rac1, and Cdc42Hs) were substrates for ToxA, whereas H-Ras, Rab5, and Arf1 were not glucosylated. ToxA-catalyzed glucosylation of lysates from ToxA-pretreated rat basophilic leukemia (RBL) cells resulted in a decreased incorporation of [14C]glucose, indicating previous glucosylation in the intact cell. Glucosylation of the Rho subtype proteins appears to be the molecular mechanism by which C. difficile ToxA mediates its cytotoxic effects on cells.

Amino Acids↗
PubMed2003

Recurrent Clostridium difficile colitis: case series involving 18 patients treated with donor stool administered via a nasogastric tube.

Clostridium difficile-associated diarrhea and colitis have emerged as major complications associated with use of systemic antimicrobials. In this study, the medical records for 18 subjects who received donor stool by nasogastric tube for recurrent C. difficile infection during a 9-year period at a single institution were retrospectively reviewed. During the period between the initial diagnosis of C. difficile colitis and the stool treatments, the 18 subjects received a total of 64 courses of antimicrobials (range, 2-7 courses; median, 3 courses). During the 90 days after receipt of treatment with stool, 2 patients died of unrelated illnesses. One of the 16 survivors experienced a single recurrence of C. difficile colitis during 90-day follow-up. No adverse effects associated with stool treatment were observed. Patients with recurrent C. difficile colitis may benefit from the introduction of stool from healthy donors via a nasogastric tube.

Aged↗
PubMed2004

A hospital outbreak of Clostridium difficile disease associated with isolates carrying binary toxin genes.

INTRODUCTION: The binary toxin genes cdt and cdtB have been detected in approximately 5% of Clostridium difficile strains. Severe C. difficile disease (CDD) may be associated with strains that carry the binary toxin genes. METHODS: From April 2001 through March 2002, 8 severe and 41 nonsevere cases of nosocomial CDD were studied. Severe cases of CDD were defined by the presence of >or=2 of the following criteria: (1) abdominal pain, (2) a white blood cell count of >20,000 or <1500 cells/mm(3), and (3) ileus or bowel wall thickening with ascites. Underlying disease was assessed by 2 methods: a modified Horn score and the presence of comorbid conditions. The presence of cdtA, cdtB, and the toxin A and toxin B genes was determined, and molecular subtyping was performed. RESULTS: All strains were positive for the toxin A and B genes, and 65.3% of the strains carried the cdtA and cdtB genes. Strains that carried the binary toxin genes accounted for 87.5% of the cases of severe CDD and 61.0% of the nonsevere cases (P=.23). Severity of CDD was not associated with either severe underlying disease or comorbid conditions. The strains that caused severe CDD belonged to 4 protein profile groups and >or=3 restriction endonuclease analysis (REA) groups. All (i.e., 5 of 5) strains in REA group BI, compared with none (i.e., 0 of 7) of the strains in REA group J carried the binary toxin genes (P=.001). Strains that belonged to REA groups BK and BR also carried the binary toxin genes. CONCLUSIONS: The binary toxin genes were present in nearly two-thirds of the C. difficile strains, and they were correlated with the REA group. Severity of CDD was not closely associated with a specific clone or underlying disease, but it may be associated with the presence of the binary toxin genes. Larger studies are needed to discern whether a true association exists and whether the binary toxin alters the pathogenicity of the C. difficile strain.

ADP Ribose Transferases↗