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PubMed1992

Impaired detection of faecal verocytotoxin in the presence of Clostridium difficile cytotoxin in patients with haemolytic uraemic syndrome.

Three cases of haemolytic uraemic syndrome associated with infection with verocytotoxin producing Escherichia coli are described. The concomitant presence of Clostridium difficile cytotoxin in the patients' stool impaired the detection of free faecal verocytotoxin. Stool specimens containing Clostridium difficile cytotoxin should thus be considered negative for verocytotoxin only after neutralisation of the Clostridium difficile cytotoxin with antitoxin.

Animals↗
PubMed2000

Hospital disinfectants and spore formation by Clostridium difficile.

Evidence is lacking on how best to decontaminate the hospital environment of Clostridium difficile. We compared sporulation levels in the UK epidemic C. difficile strain (P24), another clinical isolate (B31), and an environmental strain (E4) cultured in faecal emulsion containing subinhibitory concentrations of one of five hospital cleaning agents. The epidemic strain produced significantly more spores than the non-prevalent strains, and sporulation was further enhanced when this strain was cultured in faeces exposed to non-chlorine-based cleaning agents. The choice of cleaning agent can have a substantial effect on the persistence of C. difficile spores in the hospital environment.

Analysis of Variance↗
PubMed2000

Analysis of the pathogenicity locus in Clostridium difficile strains.

The genes for Clostridium difficile toxins A and B (tcdA and tcdB) are part of a 19.6-kb pathogenicity locus (PaLoc) that includes the genes tcdD, tcdE, and tcdC. To determine whether the C. difficile PaLoc is a stable and conserved genetic unit in toxigenic strains, a multiplex polymerase chain reaction was used to analyze 50 toxigenic, 39 nontoxigenic, and 2 toxin-defective isolates. The respective amplicons were identified for tcdA-E in the toxigenic isolates; these were absent in the nontoxigenic isolates. C. difficile P-829 lacked at least a fragment of tcdD, tcdB, tcdE, and tcdC, but tcdA was present. C. difficile 8864 had deletions in the tcdA and tcdC genes. These data suggest that the PaLoc is highly stable in toxigenic C. difficile, nontoxigenic isolates lack the unit, and isolates with a defective PaLoc can still cause clinical disease. Further studies are needed to define the role of individual genes in the pathogenesis of C. difficile-associated diarrhea.

Bacterial Proteins↗
PubMed1998

Treatment of Clostridium difficile infection.

The treatment options for Clostridium difficile infection remain limited, although promising agents are currently being assessed. Metronidazole is the first-line drug of choice for those patients requiring specific anti-C. difficile treatment. Much of the interest in alternative therapies has centred on the difficult management issues posed by patients with multiple symptomatic recurrences of C. difficile infection. However, it is now clear that the majority of these episodes are due to reinfections with new C. difficile strains and not relapses caused by the original bacterium. Hence, the true efficacy of the alternative regimens remains unclear. Individuals susceptible to C. difficile reinfections need to be protected from exposure to C. difficile until their bowel flora recovers. While several biotherapeutic approaches to the treatment and prevention of C. difficile infection have been described, few controlled data are available. Preliminary studies with anti-C. difficile bovine immunoglobulin concentrates for treatment and prevention have produced promising results. Vaccination to prevent C. difficile infection, particularly in high-risk elderly patients managed within institutions where C. difficile is endemic, is a worthwhile therapeutic goal.

Animals↗
PubMed1996

Enhancement of Clostridium difficile toxin production in biotin-limited conditions.

The effect of biotin on toxin production by Clostridium difficile was examined in a defined medium. When toxin production by strain KZ 1647, which was isolated from a healthy adult, was examined in relation to its biotin requirement, it was found that with decreasing concentrations of biotin, bacterial growth was decreased, but production of both toxins A and B were remarkably increased, particularly with 0.05 nM biotin. The time course of production of both toxins in biotin-limited conditions was similar to that in biotin-enriched conditions. The biotin effect on toxin production was also observed in 15 other strains, suggesting that the effect occurs frequently amongst toxigenic C. difficile strains. The biotin effect is discussed in relation to the pathogenesis of C. difficile colitis.

Bacterial Proteins↗
PubMed2006

The mosaic nature of intergenic 16S-23S rRNA spacer regions suggests rRNA operon copy number variation in Clostridium difficile strains.

Clostridium difficile is a major spore-forming environmental pathogen that causes serious health problems in patients undergoing antibiotic therapy. Consequently, reliable and sensitive methods for typing individual strains are required for epidemiological and environmental studies. Ribotyping is generally considered the best method, but it fails to account for sequence diversity which might exist in intergenic 16S-23S rRNA spacer regions (ISRs) within and among strains of this organism. Therefore, this study was undertaken to compare the sequence of each individual ISR in five strains of C. difficile to explore the extent of this diversity and see whether such information might provide the basis for more sensitive and discriminatory strain typing methods. After targeted PCR amplification, cloning, and sequencing, the diversity of the ISRs was used as a measure of rRNA operon copy number. In C. difficile strains 630, ATCC 43593, A, and B, 11, 11, 7, and 8 ISR length variants, respectively, were found (containing different combinations of sequence groups [i to xiii]), suggesting 11, 11, 7, and 8 rrn copies in the respective strains. Many ISRs of the same length differed markedly in their sequences, and some of these were restricted in occurrence to a single strain. Most of these ISRs did not contain any tRNA genes, and only single copies of the tRNA(Ala) gene were found in those that did. The presence of ISR sequence groups (i to xiii) varied between strains, with some found in one, two, three, four, or all five strains. We conclude that the intergenic 16S-23S rRNA spacer regions showed a high degree of diversity, not only among the rrn operons in different strains and different rrn copies in a single strain but also among ISRs of the same length. It appears that C. difficile ISRs vary more at the inter- and intragenic levels than those of other species as determined by empirical comparison of sequences. The precise characterization of these sequences has demonstrated a high level of mosaic sequence block rearrangements that are present or absent in multiple strain-variable rrn copies within and between five different strains of C. difficile.

Bacterial Typing Techniques↗
PubMed2001

Clostridium beijerinckii and Clostridium difficile detoxify methylglyoxal by a novel mechanism involving glycerol dehydrogenase.

In contrast to gram-negative bacteria, little is known about the mechanisms by which gram-positive bacteria degrade the toxic metabolic intermediate methylglyoxal (MG). Clostridium beijerinckii BR54, a Tn1545 insertion mutant of the NCIMB 8052 strain, formed cultures that contained significantly more (free) MG than wild-type cultures. Moreover, BR54 was more sensitive to growth inhibition by added MG than the wild type, suggesting that it has a reduced ability to degrade MG. The single copy of Tn1545 in this strain lies just downstream from gldA, encoding glycerol dehydrogenase. As a result of antisense RNA production, cell extracts of BR54 possess significantly less glycerol dehydrogenase activity than wild-type cell extracts (H. Liyanage, M. Young, and E. R. Kashket, J. Mol. Microbiol. Biotechnol. 2:87-93, 2000). Inactivation of gldA in both C. beijerinckii and Clostridium difficile gave rise to pinpoint colonies that could not be subcultured, indicating that glycerol dehydrogenase performs an essential function in both organisms. We propose that this role is detoxification of MG. To our knowledge, this is the first report of targeted gene disruption in the C. difficile chromosome.

Biodegradation, Environmental↗
PubMed2002

Molecular analysis of the pathogenicity locus and polymorphism in the putative negative regulator of toxin production (TcdC) among Clostridium difficile clinical isolates.

The pathogenicity locus (PaLoc) of Clostridium difficile contains toxin A and B genes and three accessory genes, including tcdD and tcdC, which are supposed to code for the positive and negative regulators of toxin expression, respectively. Different studies have described variations in C. difficile toxin A and B genes, but little is known about C. difficile variants for the accessory genes. The PaLoc of several C. difficile clinical isolates was investigated by three different PCR methods with the aim to identify variant strains. Of the toxinogenic C. difficile strains examined, 25% showed variations. No correlation between C. difficile variant strains and key patient groups was found. Interestingly, all of these strains showed a variant tcdC gene. Three different tcdC alleles were identified, and one of these had a nonsense mutation which reduced the TcdC protein from 232 to 61 amino acids. It is possible that different TcdC variants affect toxin production differently, a hypothesis with important implications for the pathogenic potential of variant C. difficile strains.

Amino Acid Sequence↗
PubMed2005

Frequency of sample submission for optimal utilization of the cell culture cytotoxicity assay for detection of Clostridium difficile toxin.

We reviewed the results of repeated sample submissions within a 7-day time frame for Clostridium difficile toxin testing. A total of 2,940 samples were tested during a 3-month period using a cell culture cytotoxicity assay (CCCA). The results from all second samples (n = 1,101) were concordant with the original test result. In only two cases (0.8%; n = 247) was a third sample positive when the first two samples were negative. In this study, submission of multiple samples for CCCA did not increase detection of Clostridium difficile infection.

Bacterial Toxins↗
PubMed2004

Isolation of non-toxigenic strains of Clostridium difficile from cases of diarrhea among patients hospitalized in hematology/oncology ward.

Clostridium difficile has become the most common cause of hospital acquired diarrhea after antibiotic treatment. The aim of this study was to determine the frequency of C. difficile associated diarrhea among hematology/oncology ward patients and to characterize isolated strains. Twenty three toxigenic and thirteen non-toxigenic strains were detected among fecal isolates. Antibiotic susceptibility testing to erythromycin and clindamycin demonstrated a high degree of resistance (MIC > 256 ug/ml) to both antibiotics in 9 out of 13 nontoxigenic C. difficle strains. Out of 7 patients with maximal frequency of diarrhea (10 empties/day) in 4 cases non-toxigenic strains of C. difficile were isolated. In these cases duration of diarrhea was longer in time than in cases of diarrhea caused by toxigenic strains. Further investigation with a larger patient population is necessary to better understand the role that non-toxigenic C. difficile strains play in disease development.

Clostridioides difficile↗
PubMed1990

Incidence of cytotoxin producing isolates of Clostridium difficile in faeces of neonates and children in Nigeria.

One hundred and fifty six (156) confirmed isolates of Clostridium difficile from faeces of neonates and children in parts of Anambra State, Nigeria were screened and assayed for cytotoxin production by the tissue culture technique and the frequency of occurrence estimated. Twenty three out of 156 isolates were found to be cytotoxin positive isolates representing a frequency of 14.8%. There was no difference between the frequency of occurrence of cytotoxin positive isolates in neonates and children from rural and urban areas. Infants in the age group of one dy to 1 yr showed 16.7% frequency of occurrence of cytotoxin positive isolates, with toxin titers between 5 to 1280, 10% for children of 1-2 yrs age group, with titers between 5 to 40 and 8.3% for children of 2 to 3 yrs age group with titers of 5 and 10. No cytotoxin positive isolate was detected from children of the 3 to 5 yrs age group. Children fed by formula foods alone showed a 50% frequency of occurrence of cytotoxin positive isolates, children fed by breast milk plus formula supplementation, 19.23% and breast milk alone 17.5%. There was no significant difference in the frequency of occurrence of cytotoxin positive isolates in faeces from diarrhoeal and non diarrhoeal cases. It appears therefore, that age and mode of feeding are important factors that influence intestinal colonization of cytotoxin producing isolates of C. difficile in neonates and children.

Age Factors↗