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PubMed2001

Molecular and genomic analysis of genes encoding surface-anchored proteins from Clostridium difficile.

The gene slpA, encoding the S-layer precursor protein in the virulent Clostridium difficile strains C253 and 79--685, was identified. The precursor protein carries a C-terminal highly conserved anchoring domain, similar to the one found in the Cwp66 adhesin (previously characterized in strain 79--685), an SLH domain, and a variable N-terminal domain mediating cell adherence. The genes encoding the S-layer precursor proteins and the Cwp66 adhesin are present in a genetic locus carrying 17 open reading frames, 11 of which encode a similar two-domain architecture, likely to include surface-anchored proteins.

Amino Acid Sequence↗
PubMed2000

Genetic characterization of toxin A-negative, toxin B-positive Clostridium difficile isolates by PCR.

Toxin-specific enzyme immunoassays, cytotoxicity assays, and PCR were used to analyze 48 toxin A-negative, toxin B-positive Clostridium difficile isolates from various geographical sites around the world. All the isolates were negative by the TOX-A TEST and positive by the TOX A/B TEST. A deletion of approximately 1.7 kb was found at the 3' end of the toxA gene for all the isolates, similar to the deletion in toxinotype VIII strains (e.g., C. difficile serotype F 1470). Additional PCR analysis indicated that the toxin B encoded by these isolates contains sequence variations downstream of the active site compared to the sequence of reference strain VPI 10463. This variation may extend the glucosylation spectrum to Ras proteins, as observed previously for closely related lethal toxin from Clostridium sordellii and toxin B from toxin A-negative, toxin B-positive strain F 1470. Toxin A-negative, toxin B-positive isolates have recently been associated with disease in humans, and they may be more common than was previously supposed.

Bacterial Proteins↗
PubMed1994

Clostridium difficile in antibiotic associated pediatric diarrhea.

A case control study was carried out at the medical wards of Dr. B.C. Roy Memorial Hospital for Children, Calcutta, between January and September 1989. One hundred eleven hospitalized children up to the age of 5 years, receiving antibiotics for different medical problems, developed antibiotic associated diarrhea. Isolation of Clostridium difficile as sole pathogen was very low (3.6%) from these patients. Fecal samples of 111 case matched control children were also screened for C.difficile. Only 2.7% fecal samples of control children were positive for C.difficile. All the strains of C.difficile isolated from antibiotic associated diarrhea cases showed neutralisable cytotoxin in in vitro test. In contrast none of the strains isolated from control children showed cytotoxicity. This study suggests that C.difficile is not an important pathogen related to antibiotic associated diarrhea in children at this hospital.

Anti-Bacterial Agents↗
PubMedDate not supplied

The roles of Clostridium difficile and enterotoxigenic Clostridium perfringens in diarrhea in dogs.

In this prospective study, feces of dogs with diarrhea were compared with feces of normal dogs for the presence of Clostridium difficile, C difficile toxins A and B, C perfringens, and C perfingens enterotoxin (CPE). C difficile toxins A, B, or both were present in feces of 18 of 87 (21%) dogs with diarrhea and 4 of 55 (7%) normal dogs (P = 0.03), whereas CPE was present in the feces of 24 of 87 (28%) dogs with diarrhea and 3 of 55 (5%) normal dogs (P = 0.01). C difficile was isolated from 2 of 87 (2%) dogs with diarrhea but was not isolated from the feces of 55 normal dogs, possibly because of poor survival of the organism in fecal samples. C perfringens was isolated from the feces of 23 of 24 (96%) CPE-positive dogs with diarrhea, 52 of 63 (83%) CPE-negative dogs with diarrhea, and 39 of 55 (71%) CPE-negative dogs with normal feces. No correlation was found between C perfringens spore number and the presence of CPE.

Animals↗
PubMed2005

[Clostridium difficile ribotype 027, toxinotype III in The Netherlands].

Recently, two Dutch hospitals reported outbreaks of Clostridium difficile ribotype 027, toxinotype III. This strain, which was seen earlier in the United States, Canada and the United Kingdom, produces large amounts of toxins due to a defect in the toxin-regulating gene and causes severe diarrhoea. Antibiotic use, especially use of fluoroquinolones, is a risk factor. Control of outbreaks is hampered by the fact that Clostridium forms spores that can survive for a very long time in the environment and are resistant to the usual surface disinfectants. Protocols for diagnostic investigations, prevention and control of outbreaks are available.

Bacterial Toxins↗
PubMed1999

Trends in infection morbidity in a pediatric oncology ward, 1986-1995.

BACKGROUND AND PROCEDURE: We retrospectively studied the type, severity, frequency, and outcome of febrile infectious complications in 217 cancer patients receiving cytotoxic chemotherapy (603 episodes) over a 10-year period in a single pediatric institution. RESULTS: A total of 48.8% of the episodes occurred in severely leukopenic patients (WBC < 1.0 x 10(9)/l, absolute neutrophil count < 500 x 10(6)/l). In the second half of the study period febrile episodes occurred at increased frequency. The number of patients with gram-positive isolates in blood cultures increased over the years, most frequently coagulase-negative staphylococci were found. Remarkably, gram-negative bacteria increasingly resistant to the administered first-line antibiotic regimen emerged, necessitating modifications of the antimicrobial strategy every 3 years. Furthermore, Clostridium difficile-associated enterocolitis posed a clinical problem at increasing frequency since 1993. As expected, the speed of leukocyte recovery within 5 days from the onset of a febrile complication had an influence on the outcome of these episodes. CONCLUSIONS: Rapid recovery of the WBC was associated with an excellent prognosis whereas persisting neutropenia was found to be a negative factor associated with fatal outcomes. The fatality rate of all febrile episodes (2.3%) remained the same throughout the study period despite the availability and wider use of recombinant hematopoietic growth factors since 1991.

Adolescent↗
PubMed2006

Distribution of Clostridium difficile PCR ribotypes in regions of Hungary.

The objective of this survey was to determine the distribution of Clostridium difficile PCR ribotypes present across three Hungarian geographical regions. A total of 105 isolates of C. difficile from diarrhoeal faeces of both inpatients and outpatients were examined. The toxigenic status of the strains was determined by PCR for the tcdA, tcdB, cdtA and cdtB genes in Szeged (Hungary), while strains were subjected to PCR ribotyping in Cardiff (UK). A total of 31 ribotypes were detected among the 105 C. difficile isolates tested. Five PCR ribotypes were distinct from all previously described types, suggesting that they are new. The most common types in Hungary, during the period examined, were PCR ribotype 014 (24.8 %) and PCR ribotype 002 (13.3 %). The distribution of PCR ribotypes differed in the various Hungarian regions: PCR ribotype 012 was frequent (20.7 %) in South Hungary, whereas this type was rare in the Budapest region and was not common to West Hungary. In West Hungary and the Budapest region, PCR ribotype 014 was most frequent (28.9 and 29 %, respectively).

ADP Ribose Transferases↗
PubMed1999

PCR targeted to the 16S-23S rRNA gene intergenic spacer region of Clostridium difficile and construction of a library consisting of 116 different PCR ribotypes.

A reference library of types of Clostridium difficile has been constructed by PCR ribotyping isolates (n = 2,030) from environmental (n = 89), hospital (n = 1,386), community practitioner (n = 395), veterinary (n = 27), and reference (n = 133) sources. The library consists of 116 distinct types identified on the basis of differences in profiles generated with PCR primers designed to amplify the 16S-23S rRNA gene intergenic spacer region. Isolates from 55% of infections in hospitals in the United Kingdom belonged to one ribotype (type 1), but this type was responsible for only 7. 5% of community infections.

Animals↗
PubMed2004

Clostridium difficile IStron CdISt1: discovery of a variant encoding two complete transposase-like proteins.

Screening a Clostridium difficile strain collection for the chimeric element CdISt1, we identified two additional variants, designated CdISt1-0 and CdISt1-III. In in vitro assays, we could prove the self-splicing ribozyme activity of these variants. Structural comparison of all known CdISt1 variants led us to define four types of IStrons that we designated CdISt1-0 through CdISt1-III. Since CdISt1-0 encodes two complete transposase-like proteins (TlpA and TlpB), we suggest that it represents the original genetic element, hypothesized before to have originated by fusion of a group I intron and an insertion sequence element.

Clostridioides difficile↗
PubMed2006

[Pseudomembraneous colitis caused by a toxin B-positive and a toxin A-negative strain of Clostridium difficile].

We describe a case of pseudomembraneous colitis (PMC) caused by a toxin A- B+ strain of Clostridium difficile (CD). In Denmark only a few laboratories investigate for toxin production, and if they do, the toxin A enzyme immunoassay (EIA) is the test generally used when testing for CD. This toxin A negative but toxin B positive strain thus remains undetectable. If CD-associated diarrhea is clinically suspected and tests for toxin A are negative, infection with a toxin A- B+ strain should be considered. Further diagnostic tests such as cellular cytotoxicity assays or toxin A/B EIA should be performed. The standard treatment of PMC in severely ill patients should be vancomycin, administered orally.

Aged↗
PubMed1997

Comparison of cycloserine-cefoxitin-fructose agar (CCFA) and taurocholate-CCFA for recovery of Clostridium difficile during surveillance of hospitalized patients.

The effectiveness of cycloserine-cefoxitin-fructose agar (CCFA) and taurocholate-CCFA (TCCFA) in isolating Clostridium difficile from swabs of the rectum or stools from 184 hospitalized patients who were monitored weekly and when they had diarrhea was compared. The number of surveillance time points ranged from two to eight per patient over a period of 4 to 34 days per patient, totalling 621 comparisons of the media. C. difficile was isolated more frequently by TCCFA than CCFA at seven of eight surveillance points, a significant trend (O'Brien test, p = 0.002). This difference reached statistical significance at the second surveillance time point when the prevalence of C. difficile was sufficiently high. At the second surveillance point, C. difficle was isolated only by TCCFA in 7 of 184 comparisons of the media, only by CCFA in none of the comparisons, and by both media in 19 comparisons (p = 0.016). C. difficle was first isolated at an earlier surveillance time point on TCCFA in 11 of 36 patients and on CCFA first only once (p = 0.005). Use of TCCFA media increased the rapidity and sensitivity of culture for C. difficle when doing patient surveillance but did not increase sensitivity when diagnosing patients with diarrhea.

Antibiotics, Antitubercular↗
PubMed1992

Influence of radiotherapy on intestinal microflora in cancer patients.

We investigated in 15 patients with carcinoma of the uterine cervix or endometrium, who were undergoing postoperative radiation therapy, the effects of different fractionated radiation exposures on counts of fecal bacteria, on the growth of Clostridium difficile and Clostridium perfringens enterotoxin production. We observed a generally significant decrease in intestinal microflora after the first radiation exposure, whereas at the end of radiotherapy all bacteria increased and reached basal values except Enterococcus faecium 1, lactobacilli and total anaerobes. In some patients we observed an overgrowth of some Clostridium spp. which were potential pathogens associated with clinical symptoms. We did not observe an influence of multiple radiations on C. perfringens enterotoxin fecal contents. We conclude that patients receiving radiotherapy may benefit from the intake of oral bacteriotherapy, i.e. live beneficial bacteria such as Bacillus subtilis at the beginning of the irradiation exposure.

Aged↗
PubMed2000

Characterization of the ends and target sites of the novel conjugative transposon Tn5397 from Clostridium difficile: excision and circularization is mediated by the large resolvase, TndX.

Tn5397 is a conjugative transposon that was originally isolated from Clostridium difficile. Previous analysis had shown that the central region of Tn5397 was closely related to the conjugative transposon Tn916. However, in this work we obtained the DNA sequence of the ends of Tn5397 and showed that they are completely different to those of Tn916. Tn5397 did not contain the int and xis genes, which are required for the excision and integration of Tn916. Instead, the right end of Tn5397 contained a gene, tndX, that appears to encode a member of the large resolvase family of site-specific recombinases. TndX is closely related to the TnpX resolvase from the mobilizable but nonconjugative chloramphenicol resistance transposons, Tn4451 from Clostridium perfringens and Tn4453 from C. difficile. Like the latter elements, inserted copies of Tn5397 were flanked by a direct repeat of a GA dinucleotide. The Tn5397 target sites were also shown to contain a central GA dinucleotide. Excision of the element in C. difficile completely regenerated the original target sequence. A circular form of the transposon, in which the left and right ends of the element were separated by a GA dinucleotide, was detected by PCR in both Bacillus subtilis and C. difficile. A Tn5397 mutant in which part of tndX was deleted was constructed in B. subtilis. This mutant was nonconjugative and did not produce the circular form of Tn5397, indicating that the TndX resolvase has an essential role in the excision and transposition of Tn5397 and is thus the first example of a member of the large resolvase family of recombinases being involved in conjugative transposon mobility. Finally, we showed that introduction of Tn916 into a strain containing Tn5397 induced the loss of the latter element in 95.6% of recipients.

Amino Acid Sequence↗
PubMedDate not supplied

Contributions to the taxonomy and biology of Clostridium difficile.

Clostridium difficile was incriminated by Hughes and Jarvis (1987) as a cause of intestinal infections in USA in the 1980-1984 period in 45 p. 100 of cases, whereas Salmonellae only in 12 p. 100. Four strains of this organism are studied in this paper in comparison with ten strains of C. bifermentans and six of C. sordellii, because the three species share a common antigen and have other common characteristics, as well. However, spores of C. difficile swell the bacteria and those of other bacteria (C. bifermentans and C. sordellii) do not; C. difficile does not produce indole, whereas the other species produce it. We confirmed the selective capacity of the medium of George et al. (1979) using the "alcohol shock" and as selective agents cycloserine D and cefoxitin. C. difficile proved to be most susceptible to metronidazole and rifampin. Whereas the former antibiotic was considered as a cause of post-antibiotic intestinal infections by different authors, the second was not, to our knowledge. The strain 10463 has a considerable toxicity (1000 DLM/ml for the white mouse, and pathogenicity--2000--5000 DCL for the white mouse, as compared to 25 DCL of the other three strains). Using this toxin an antitoxic serum was obtained in horse, with a capacity of neutralizing the action of the toxin up to a dilution of 1 p. 1000.

Aerobiosis↗
PubMed1994

Etiology and management of toxic megacolon in patients with human immunodeficiency virus infection.

We report six cases of toxic megacolon in patients with human immunodeficiency virus (HIV). One case, at an early stage of HIV infection, mimicked a severe attack of Crohn's disease, with a negative search for infectious agents. Subtotal colectomy was successfully performed with an uneventful postoperative course. The five other cases concerned patients with acquired immunodeficiency syndrome at a late stage of immunodeficiency. They were related to Clostridium difficile or cytomegalovirus (CMV) intestinal infection in two and three patients, respectively. One case of CMV colitis presented macroscopically and histologically as pseudomembranous colitis. Emergency subtotal colectomy, performed in the first four patients with acquired immunodeficiency syndrome was followed by a fatal postoperative outcome. The last patient treated conservatively by colonoscopic decompression, in association with anti-CMV therapy, had a favorable short-term outcome. From the experience of our series and data from the literature, we discuss the best diagnostic and therapeutic approach to toxic megacolon in patients with HIV.

Adult↗
PubMed2006

Moxifloxacin prophylaxis in neutropenic patients.

OBJECTIVES: Recent studies have shown a beneficial impact of fluoroquinolones on infection-related morbidity and mortality for patients with haematological malignancies during neutropenia. Among the fluoroquinolones moxifloxacin currently provides one of the broadest spectra of antibacterial activity and may be suitable for prophylaxis during neutropenia. PATIENTS AND METHODS: In this controlled before and after observational study, moxifloxacin chemoprophylaxis was used during prolonged neutropenia in haemato-oncological patients (period 2; 282 episodes). Data were compared with two periods with levofloxacin prophylaxis, one preceding period (period 1; 399 episodes) and a post-observational period (period 3; 53 episodes). Endpoints for evaluation were the rates of Gram-negative and Gram-positive bacteraemia and infection-related mortality. RESULTS: We found similar survival rates as compared with levofloxacin. The rate of Gram-negative bacteraemia was higher during prophylaxis with moxifloxacin (11%) when compared with levofloxacin (6% for period 1 and 6% for period 3). In addition we observed a marked increase in diarrhoea associated with Clostridium difficile toxin A (CDAD) after a formula change from levofloxacin to moxifloxacin (attack rate 6% versus 33%). A decrease was attained after changing back to levofloxacin and reinforcing hygienic measures (13%). CONCLUSIONS: During moxifloxacin prophylaxis, we observed a significantly increased incidence of Gram-negative bacteraemia and CDAD. Careful attention must be paid not to trade the particularly beneficial effects of fluoroquinolones in the neutropenic setting for such disadvantageous effects. Until further data are obtained, caution is warranted when applying fluoroquinolones with high anaerobic activity in the neutropenic setting.

Anti-Infective Agents↗
PubMedDate not supplied

In vitro activity of rifaximin, metronidazole and vancomycin against Clostridium difficile and the rate of selection of spontaneously resistant mutants against representative anaerobic and aerobic bacteria, including ammonia-producing species.

BACKGROUND: Rifaximin is a rifamycin derivative characterized by a wide antibacterial activity. This drug is neither absorbed by the gastrointestinal tract nor inactivated by gastric juices, and exerts its action entirely within the intestinal lumen. METHODS: In this study, the activity of this antibiotic was compared with that of metronidazole and vancomycin against 93 Clostridium difficile isolates. The rate of emergence of bacteria spontaneously resistant to the new compound was also evaluated in relation to representative gram-positive and gram-negative strains. In terms of MIC(50) values, rifaximin showed an intrinsic activity superior to that of the other agents. The emergence of spontaneously resistant strains was assessed with 46 aerobic (staphylococci, enterococci, Proteus spp., Citrobacter freundii, Providencia rettgeri, enteropathogenic, enteroinvasive, enterotoxigenic and entero- hemorrhagic Escherichia coli, and Salmonella enteritidis) and anaerobic (Clostridium spp., Bacteroides spp., Fusobacterium nucleatum and Peptococcus spp.) pathogens, most of them also ammonium producers. Two different methods, broth and agar dilution, were employed. RESULTS: When liquid medium was employed, bacteria capable of sustained growth in 100 microg/ml of rifaximin were obtained after 2-5 transfers with gram-positive aerobic cocci, 2-3 transfers with gram-negative aerobic strains and 2-5 transfers with anaerobic species. At the highest dose used with the agar dilution method (8 x MIC), the frequency of emergence of spontaneously resistant mutants ranged from <1 x 10(-9) to 1.6 x 10(-8) with gram-positive aerobic and anaerobic cocci, while with aerobic and anaerobic gram-negative bacteria, this value ranged from <1 x 10(-9) to 1.7 x 10(-7). C. difficile showed a particularly low incidence of spontaneously resistant mutants (<1 x 10(-9)). The low incidence of resistant subpopulations selected by levels of 8 x MIC of rifaximin suggests that the high levels of the drug which were reached in the gastrointestinal lumen may further prevent the selection of mutants. CONCLUSION: The low toxicity, broad antibacterial activity and very poor absorption from the gastrointestinal tract of rifaximin suggest a potential therapeutic use for this drug in gastrointestinal diseases, as well as in the management of patients with cirrhosis and chronic portal-systemic encephalopathy.

Ammonia↗
PubMed2002

Evaluation of a routine diagnostic fecal panel for dogs with diarrhea.

OBJECTIVES: To assess the diagnostic yield of a routine fecal panel and determine whether Clostridium perfringens or C difficile toxin production is associated with acute hemorrhagic diarrheal syndrome (AHDS) in dogs. DESIGN: Case-control study. ANIMALS: 260 dogs with diarrhea and 177 dogs with normal feces. PROCEDURE: Medical records were reviewed for results of culture for C difficile, Campylobacterspp, and Salmonella spp; C perfringens fecal enterotoxin (CPE) assay via ELISA or reverse passive latex agglutination (RPLA) assay; fecal endospore enumeration; C difficile toxin A assay; and parasite evaluation. RESULTS: Prevalence of CPE in dogs with diarrhea was 22/154 (14.3%) via ELISA and 47/104 (45.2%) via RPLA assay, versus 9/74 (12%) via ELISA and 26/103 (25%) via RPLA assay in control dogs. Prevalence of C difficile was 47/260 (18%) in dogs with diarrhea and 41/74 (55%) in control dogs. Prevalence of C difficile toxin A was 26/254 (10.2%) in dogs with diarrhea and 0/74 in control dogs. Diagnosis of AHDS was made in 27 dogs; 8 had positive results for CPE, 7 had positive results for toxin A, and 1 had positive results for both toxins. Campylobacter spp were isolated from 13 of 260 (5%) dogs with diarrhea and 21 of 74 (28.4%) control dogs. Salmonella spp were isolated from 3 (1.2%) dogs with diarrhea. CONCLUSIONS AND CLINICAL RELEVANCE: Diagnostic value of a fecal panel in dogs with diarrhea appears to be low.

Agglutination Tests↗