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PubMed2006

High-level resistance to moxifloxacin and gatifloxacin associated with a novel mutation in gyrB in toxin-A-negative, toxin-B-positive Clostridium difficile.

OBJECTIVES: To determine the mechanism of high-level resistance to fluoroquinolone antimicrobials in toxin-A-negative, toxin-B-positive (A- B+) Clostridium difficile isolates. METHODS: Following culture 16-23S PCR ribotyping was used to determine genomic relationships between A- B+ C. difficile isolates. Antimicrobial susceptibilities were determined using Etests in the presence and absence of the efflux pump inhibitors reserpine (20 microg/mL), L-phenylalanine-L-arginine-beta-naphthylamide (PAbetaN; 20 microg/mL) and verapamil (100 microg/mL). Genomic regions including the quinolone-resistance-determining-region (QRDR) of gyrA and gyrB were amplified and characterized. RESULTS: PCR ribotyping profiles identified one major cluster of A- B+ C. difficile, universally resistant to the fluoroquinolones tested (ofloxacin, ciprofloxacin, levofloxacin, moxifloxacin and gatifloxacin; MICs > 32 mg/L). All isolates with high-level resistance had a transversion mutation (A-->T) resulting in the amino acid substitution Asp-426-->Val in gyrB. Non-clonal isolates were susceptible to moxifloxacin and gatifloxacin (MICs 0.3 and 0.4 mg/L, respectively) with reduced susceptibility to levofloxacin (MIC 3 mg/L) consistent with the wild-type genotype. The MICs for resistant isolates were not significantly affected by the addition of any of the efflux pump inhibitors. No amino acid substitutions were identified in the QRDR of gyrA. CONCLUSIONS: High-level resistance to fluoroquinolones in A- B+ C. difficile is associated with a novel transversion mutation in gyrB. The emergence of universal resistance in different C. difficile strain types may be a factor promoting outbreaks in hospitals.

Amino Acid Substitution↗
PubMed2002

Proteins released during high toxin production in Clostridium difficile.

The mechanism by which toxins A and B are released by Clostridium difficile is unknown and information about the other extracellular proteins of this bacterium is limited. The authors identified exported proteins from C. difficile strain VPI 10463 during conditions promoting high toxin production. Toxins A and B were released in a 1:1 ratio and the proportion of toxin in the extracellular fraction reached 50% during the stationary phase as compared to a proportion of <1% for typical cytoplasmic proteins, showing that toxin export was not due to bacterial lysis. A 47 kDa protein, released with similar kinetics to the toxins, was processed and showed weak similarity to the channel-forming protein TolC. Another protein released during high toxin production was unprocessed and showed similarity to XkdK encoded by the prophage PBSX in Bacillus subtilis, a protein supposedly exported via phage-specific holins. The two most abundant extracellular C. difficile proteins, found during both high and low toxin production, were processed and identified as shed S-layer proteins. As shown by N-terminal sequencing and PCR-based methods, there was a considerable sequence variation of the S-layer gene slpA in different serogroup reference strains. To conclude, C. difficile uses the classical Sec-dependent and probably also holin-like pathways to secrete a comparatively small repertoire of proteins.

Amino Acid Sequence↗
PubMed2000

Toxin gene analysis of a variant strain of Clostridium difficile that causes human clinical disease.

A toxin variant strain of Clostridium difficile was isolated from two patients with C. difficile-associated disease (CDAD), one of whom died from extensive pseudomembranous colitis. This strain, identified by restriction endonuclease analysis (REA) as type CF2, was not detected by an immunoassay for C. difficile toxin A. Culture supernatants of CF2 failed to elicit significant enterotoxic activity in the rabbit ileal loop assay but did produce atypical cytopathic effects in cell culture assay. Southern hybridization, PCR amplification, and DNA sequence analyses were performed on the toxin A (tcdA) and toxin B (tcdB) genes of type CF2 isolate 5340. Type CF2 5340 tcdA exhibited a 1,821-bp truncation, due to three deletions in the 3' end of the gene, and a point mutation in the 5' end of the gene, resulting in a premature stop codon at tcdA position 139. Type CF2 5340 tcdB exhibited multiple nucleotide base substitutions in the 5' end of the gene compared to tcdB of the standard toxigenic strain VPI 10463. Type CF2 5340 toxin gene nucleotide sequences and deduced amino acid sequences showed a strong resemblance to those of the previously described variant C. difficile strain 1470, a strain reported to have reduced pathogenicity and no association with clinical illness in humans. REA of strain 1470 identified this strain as a distinct type (CF1) within the same REA group as the closely related type CF2. A review of our clinical-isolate collection identified five additional patients infected with type CF2, three of whom had documented CDAD. PCR amplification of the 3' end of tcdA demonstrated identical 1. 8-kb deletions in all seven type CF2 isolates. REA type CF2 is a toxin variant strain of C. difficile that retains the ability to cause disease in humans but is not detected in clinical immunoassays for toxin A.

Aged↗
PubMed1998

In vitro evidence that rabbit distal colonic muscularis mucosae has a Clostridium difficile toxin A receptor.

In the rabbit ileum Clostridium difficile toxin A causes inflammation and mucosal damage via a specific glycoprotein receptor that contains alpha-D-galactose. In rabbit colon toxin A also causes inflammation, and this is associated with increased myoelectric activity and eicosanoid production. The present in vitro study was undertaken to determine if a toxin A receptor on one or more layers of colonic smooth muscle could mediate the motor effects of this agent. Toxin A (20-100 microg/ml) was without effect on longitudinal and circular muscle but had two different effects on the muscularis mucosae. Initial exposure to the toxin caused increased numbers of spontaneous contractions and a small, atropine-, tetrodotoxin-, and indomethacin-resistant increase in resting tone. More importantly, however, 30-min exposure to toxin A resulted in attenuated muscularis mucosae responses to acetylcholine and K+. Both the small excitatory and the larger inhibitory effects of toxin A were abolished by pretreatment with the lectin BS-1, which binds to toxin A receptors, but not by the nonreceptor-binding lectin DBA. These data strongly suggest that toxin A causes significant motor effects on the distal colonic muscularis mucosae via a receptor-mediated mechanism. These mechanical data were supported by the presence of histologically demonstrable toxin A and BS-1 binding sites on the muscularis mucosae but not on either the longitudinal or circular muscle layers, both of which were unresponsive to the toxin. By depressing muscularis mucosae function and, ultimately, mucosal movement as a result of toxin A production, C. difficile may promote its own proliferation, thus further contributing to the development of antibiotic-associated colitis.

Acetylcholine↗
PubMed2002

Evaluation of a test for Clostridium difficile toxins A and B for the diagnosis of neonatal swine enteritis.

A commercially available 1-hour enzyme immunoassay (EIA) for detecting the presence of Clostridium difficile toxins A and B was evaluated for use in diagnosis of C. difficile infections in neonatal swine. This test was compared with a tissue culture cytotoxicity assay, which is considered to be the reference standard for the detection of C. difficile toxins. Twenty-seven samples of colonic contents and 23 fecal samples were collected from freshly euthanized neonatal swine with a history of scours. Of the 50 specimens tested, 20 were positive by the EIA test and tissue culture and 24 were negative by both tests, for an overall correlation of 88%. The sensitivity and specificity of the EIA were 91% and 86%, respectively, and the positive and negative predictive values were 84% and 86%, respectively. The EIA test is considered suitable as an aid for the diagnosis of C. difficile enteritis because of the high correlation between EIA results and those of the tissue culture cytotoxicity assay.

Animals↗
PubMed2003

[High incidence and complications of Clostridium difficile diarrhea among patients with renal diseases].

BACKGROUND: Clostridium difficile is the main agent causing antimicrobial associated nosocomial diarrhea. Chronic renal failure is a risk factor for this type of diarrhea. AIM: To study the incidence and complications of Clostridium difficile diarrhea in a university hospital and among patients with renal diseases. PATIENTS AND METHODS: Retrospective review of all cases of Clostridium difficile diarrhea that occurred in a university hospital, between June 2000 and May 2001. RESULTS: In the Nephrology Unit, 48 episodes of Clostridium difficile diarrhea occurred in 35 patients (7 cases per 100 discharges/year). This figure is higher than the global incidence in the hospital (0.53 cases per 100 discharges/year, p < 0.001). The mean age of the 33 patients with renal diseases was 63 years old and 17 of them were female. Their main diagnoses were chronic renal failure in hemodialysis in 48%, uremic syndrome in 36% and renal transplant in 6%. Seventy nine percent had a history of antimicrobial use (42% quinolones and 36% cephalosporins). In 3 patients, the only risk factor was chronic renal failure. Seventy five percent responded to metronidazole and in 27%, diarrhea recidivated, compared with a 6% recurrence rate in other units, p < 0.02). Eight patients died during hospital stay. CONCLUSIONS: Among patients with renal diseases, Clostridium difficile is frequent and associated with a high recurrence rate and mortality. Chronic renal failure may be a risk factor for its development.

Anti-Bacterial Agents↗
PubMedDate not supplied

Basic and clinical aspects of Clostridium difficile colitis.

Clostridium difficile, a gram-positive anaerobic bacillus dubbed as the difficult clostridium because it resisted early attempts of isolation and culture. After some decades in the darkness, it became famous, when in 1978, a cytotoxin of the C. difficile was found the responsible of the pseudomembranous colitis. We review in this paper aspects of the epidemiology of the C. difficile in health and disease. Also the importance of C. difficile as a cause of nosocomial infections. We review the characteristics of the toxins A and B produced by the pathogenic strains of C. difficile. Finally, clinical aspects of infection with C. difficile in special in the pseudomembranous colitis. The diagnosis, medical therapy, complications and surgical indications are briefly described.

Anti-Bacterial Agents↗
PubMed1990

[Occurrence of Clostridium difficile in feces of children with dysfunction of the digestive tract and other disorders].

Thousand and six hundred ninety two fecal samples from children of few weeks old up to over ten years were tested for the presence of Clostridium difficile. Most of them were treated with antibiotics and showed diarrhea symptoms. Hundred and twenty three strains of C-difficile were submitted to serological typing and their sensitivity to 10 selected antibiotics and chemotherapeutic agents was determined. Among 109 strains of C. difficile tested for enterotoxin production by latex test 82 strains (75.2%) were positive. Almost half (48%) of the isolated strains belonged to serotype C. Most of the strains were resistant to cefoxitin (88%) and clindamycin (76%). Over 90% of strains were sensitive to vancomycin and azlocillin and 86% to chloramphenicol and metronidazole. In the majority of patients with positive C. difficile cultures diarrhea was present, however, it was difficult to find a direct link between these symptoms and antibiotic therapy.

Adolescent↗
PubMedDate not supplied

Effect of ceftibuten on the normal intestinal microflora.

14 healthy volunteers were given 400 mg ceftibuten orally once daily for ten days. Stool specimens were collected before, during and after ceftibuten administration. Ceftibuten was well absorbed; on average 123 mg was excreted in urine 0-6 h after dosing, while only two volunteers had detectable concentrations of ceftibuten in faeces (< or = 3.2 mg/kg). There was an overgrowth of enterococci during the administration period, while the numbers of Escherichia coli and anaerobic cocci were reduced. Six volunteers were colonized by Clostridium difficile during days 4 to 17. Beta-lactamase activity was detected in faecal samples from eight volunteers and increased significantly during the administration period.

Adult↗
PubMed2000

Clostridium difficile-associated diseases. The clinical courses of 18 fatal cases.

OBJECTIVE: Severe cases of Clostridium difficile-associated diseases with sepsis seem to be rare, as are case reports about the pathogen involved and sepsis. Our objective was to investigate the frequency and the clinical courses of severe cases of C. difficile-associated diseases with a fatal outcome in our hospital. SETTING: Teaching hospital of the University of Kiel (650 beds). DESIGN: We reviewed retrospectively all deceased patients' charts who had prior histological or microbiological evidence of C. difficile-associated diarrhea (CDAD) and revised the available histological specimens of the autopsies. PATIENTS: Over a 4-year period (November 1994-October 1998) we diagnosed 304 cases of C. difficile-associated diseases in our hospital. RESULTS: Eighteen of our cases with C. difficile-associated diseases had a fatal outcome. C. difficile was not likely to be the cause of death in two of the cases. Four of the fatal infections were community-acquired and the reason for admission to the hospital. CDAD is most prevalent in elderly patients with multiple or severe underlying diseases and tends to be overlooked. Sepsis was diagnosed in 15 of our 18 patients with C. difficile-associated diseases. CONCLUSION: Our study shows that severe cases of CDAD or cases with C. difficile-associated sepsis are probably not rare. Routine testing of fecal specimens for the presence of C. difficile toxins should be considered not only in nosocomial gastrointestinal infections but also in community-acquired gastrointestinal infections of elderly people.

Adult↗
PubMed2001

Comparison of toxinotyping and PCR ribotyping of Clostridium difficile strains and description of novel toxinotypes.

Toxinotyping and PCR ribotyping are two methods that have been used to type Clostridium difficile isolates. Toxinotyping is based on PCR-RFLP analysis of a 19 kb region encompassing the C. difficile pathogenicity locus. PCR ribotyping is based on comparison of patterns of PCR products of the 16S-23S rRNA intergenic spacer region. Representative strains (101) from a C. difficile PCR ribotype library and 22 strains from previously described toxinotypes were analysed to compare ribotyping with toxinotyping. Within this panel of strains all 11 toxinotypes (0-X) described previously and an additional 5 novel toxinotypes (XI-XV) were observed. PCR ribotyping and toxinotyping correlated well and usually all strains within a given ribotype had similar changes in toxin genes. The new toxinotype XI comprises strains that did not express toxins TcdA or TcdB at detectable levels, but contained part of the tcdA gene. Strains of toxinotype XII exhibit changes only in the 5' end of the tcdB gene. Toxinotype XIV is composed of strains that have a large insertion at the beginning of the tcdA gene. A total of 25 of the 89 tested PCR ribotypes of C. difficile contained variant strains. It was estimated that they represent 7.7% of the total number of strains in the Anaerobe Reference Unit collection.

Bacterial Toxins↗
PubMed1994

Kinetics of appearance of intestinal lesions in mice mono-associated with a lethal or non-lethal strain of Clostridium difficile.

The kinetics of the appearance of intestinal lesions induced by orogastric inoculation of gnotobiotic mice with a lethal strain of Clostridium difficile (VPI) that produced toxins A and B in vivo and in vitro was studied and compared with the lesions induced by non-lethal C. difficile strain 786 that produced toxins A and B in vitro but only toxin B in measurable amounts in vivo. Different portions of the intestine were removed 12, 20, 26 and 30 h after inoculation and studied by scanning electronmicroscopy. The remaining portions were homogenised for enumeration of C. difficile and quantification of toxin A by enzyme immunoassay and toxin B by cytotoxicity. The results showed that, following inoculation: (i) measurable amounts of both toxins were necessary to produce lesions; (ii) with strain VPI, the caecum and the colon were rapidly impaired and completely destroyed after 1 day, whereas the small intestine was damaged to a lesser extent; (iii) C. difficile strain 786 did not cause mucosal damage but induced mucus-like or serum-like secretion and morphological changes in the caecal enterocytes only.

Animals↗
PubMed2007

Evaluation of real-time PCR and conventional diagnostic methods for the detection of Clostridium difficile-associated diarrhoea in a prospective multicentre study.

In this prospective multicentre study, an enzyme-linked fluorescent assay (VIDAS CDA2; bioMérieux), an enzyme-linked assay [Premier Toxins A and B (PTAB); Meridian] and an in-house real-time PCR amplifying the tcdB gene were compared with the cell cytotoxicity assay used as the 'gold standard' for diagnosis of Clostridium difficile-associated diarrhoea (CDAD). Faecal samples from patients with a request for C. difficile diagnosis and samples from patients with diarrhoea hospitalized for at least 72 h were collected for 3 consecutive months from four university medical centres in The Netherlands. In total, 547 faecal samples were obtained from 450 patients. Of 540 samples available for all of the assays, 84 (15.6 %) showed a positive result in one or more assays. The cell cytotoxicity assay was positive in 31 samples (5.7 %) from 28 patients. A diagnosis of CDAD was not considered by the physician in 5 (23.8 %) of 21 patients with CDAD who were hospitalized for at least 72 h. Compared with the cell cytotoxicity assay, the sensitivity of VIDAS, PTAB and PCR was 83.9, 96.8 and 87.1 %, respectively. The specificity of VIDAS, PTAB and PCR was 97.1, 94.3 and 96.5 %, respectively. The positive and negative predictive values for VIDAS, PTAB and PCR were 63.4 and 99.0 %, 50.9 and 99.8 %, and 60.0 and 99.2 %, respectively. Of 61 samples that were positive in one, two or three of the assays, 56 were available for discordance analysis. Discordance analysis was performed by culture of toxinogenic strains. The concordance of VIDAS, PTAB and PCR with culture was 53.6 % (30/56), 55.4 % (31/56) and 71.4 % (40/56), respectively. It was concluded that real-time PCR had the highest concordance with toxinogenic culture and is therefore the preferred method for diagnosing CDAD in faecal samples. It was also concluded that diagnosis of patients with diarrhoea who have been hospitalized for more than 72 h should focus mainly on the detection of C. difficile, irrespective of the physician's request.

Adult↗
PubMed1999

Clostridium difficile-associated diarrhea in the elderly.

OBJECTIVE: It is widely believed that Clostridium difficile (C. difficile)-associated diarrhea is a more severe disease in the elderly than in the young, associated with increased morbidity and mortality. These beliefs are largely anecdotal, and there are few data supporting them. METHODS: We conducted an evaluation in an urban, tertiary care hospital of 89 inpatients in whom C. difficile-associated diarrhea was identified. These patients were evaluated prospectively, and the group was divided by age into those < 60 yr of age (younger) and those > or = 60 yr (elderly). RESULTS: There was no difference in mortality or morbidity in elderly individuals with C. difficile-associated diarrhea when compared with younger persons similarly infected. The response to standard treatment was similar in both groups. Older patients were more likely to have an elevated white blood cell count in association with C. difficile-associated diarrhea (60% vs 26%, p < 0.05), and were more likely to have acquired their infection in the hospital (89% vs 50%, p < 0.0001). CONCLUSIONS: In the elderly, C. difficile-associated diarrhea is almost always acquired in institutions, and may not be obvious among patients' other problems. The elderly do not seem to have an increase in C. difficile diarrhea-associated morbidity or mortality. There is no evidence that C. difficile-associated diarrhea is more severe in the elderly than it is in the young.

Adolescent↗
PubMed1997

The association of erythromycin ethylsuccinate with acute colitis in horses in Sweden.

In Sweden there are several reports of mares developing acute colitis while their foals were being treated orally for Rhodococcus equi pneumonia with the combination of erythromycin and rifampicin. In this study 6 adult horses were given low oral dosages of these antibiotics, singly or in combination. Within 3 days post administration of erythromycin, in one case in combination with rifampicin, 2 horses developed severe colitis (one fatal). Clostridium difficile was isolated from one of the horses, whereas no specific pathogens were isolated from the other. Both horses had typical changes in blood parameters seen in acute colitis. Clostridium difficile was also isolated from the faeces of a third horse given an even lower dosage of erythromycin in combination with rifampicin. This horse developed very mild clinical symptoms and recovered spontaneously. In the fourth horse given erythromycin only, very high numbers of Clostridium perfringens were isolated. The horses given rifampicin only did not develop any clinical symptoms and there were no major changes in their faecal flora. In conclusion, it has been demonstrated that low dosages of erythromycin ethylsuccinate can induce severe colitis in horses associated with major changes of the intestinal microflora. Clostridium difficile has been demonstrated as a potential aetiological agent in antibiotic-induced acute colitis.

Actinomycetales Infections↗
PubMed1997

Survival of anti-Clostridium difficile bovine immunoglobulin concentrate in the human gastrointestinal tract.

To be therapeutically active, oral hyperimmune bovine immunoglobulin concentrate (BIC) must survive its passage through the intestinal tract. This led us to study the gastrointestinal stability of orally administered BIC directed against Clostridium difficile toxins (BIC-C. difficile). BIC-C. difficile was stable at neutral pH in vitro but was degraded at low pH, particularly in the presence of pepsin. Healthy volunteers (n = 6) took BIC-C. difficile (45 or 8 g) as a single oral dose. Total bovine immunoglobulin G (IgG) and specific anti-C. difficile IgG were measured in the stool. BIC was given under the following conditions: in the fasting state, in the fed state, with antacid, during omeprazole therapy, or in enteric capsules (released at pH > 6). The mean fecal bovine IgG content of 3-day stool collections was similar in the fasting (536 mg; 3.8% of the ingested dose of BIC), fed (221 mg; 1.6%), and antacid (381 mg; 2.7%) groups. Omeprazole therapy was associated with increased fecal bovine IgG levels (1253 mg; 8.8%), but this difference did not reach statistical significance (P = 0.07). Administration of 8 g of BIC-C. difficile in enteric capsules resulted in substantially higher fecal bovine IgG levels (1,124 mg; 32.7% of the oral dose) than those obtained after administration of nonencapsulated BIC (22 MG; 0.6%; P = 0.004). An inverse relationship was noted between intestinal transit time and fecal bovine IgG content (R = 0.83; P = 0.04 [data from omeprazole group]). Filtrates of stool samples collected after oral administration of BIC-C. difficile neutralized the cytotoxicity of C. difficile toxins A and B, whereas control stool filtrates did not. Bovine colostral IgG undergoes partial degradation in the intestinal tract. Exposure to acidic gastric secretions and prolonged colonic transit may both contribute to IgG degradation. Nonetheless, humans taking BIC-C. difficile orally have neutralizing antitoxin activity in their stool.

Administration, Oral↗
PubMed2003

Expression of Clostridium difficile toxins A and B and their sigma factor TcdD is controlled by temperature.

Growth temperature was found to control the expression of toxins A and B in Clostridium difficile VPI 10463, with a maximum at 37 degrees C and low levels at 22 and 42 degrees C in both peptone yeast (PY) and defined media. The up-regulation of toxin A and B mRNA and protein levels upon temperature upshift from 22 to 37 degrees C followed the same kinetics, showing that temperature control occurred at the level of transcription. Experiments with Clostridium perfringens using gusA as a reporter gene demonstrated that both toxin gene promoters were temperature controlled and that their high activity at 37 degrees C was dependent on the alternative sigma factor TcdD. Furthermore, tcdD was found to be autoinduced at 37 degrees C. Glucose down-regulated all these responses in the C. perfringens constructs, similar to its impact on toxin production in C. difficile PY broth cultures. C. difficile proteins induced at 37 degrees C and thus coregulated with the toxins by temperature were demonstrated by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and identified as enzymes involved in butyric acid production and as electron carriers in oxidation-reduction reactions. The regulation of toxin production in C. difficile by temperature is a novel finding apparently reflecting an adaptation of the expression of its virulence to mammalian hosts.

Bacterial Proteins↗