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PubMed1993

Improvement of Clostridium difficile isolation by heat-shock and typing of the isolated strains by SDS-PAGE.

Clostridium difficile plays an essential role in causing pseudomembranous colitis. We looked for the presence of these bacteria in the stools of 169 hospitalized patients and 38 nurses from wards with cases of diarrhea (207 subjects). The study was divided into three parts. In the first part, we compared three methods for isolating Clostridium difficile from stool samples: pre-selection with heat-shock, direct plating on Cycloserine-Cefotaxime-Fructose Agar (CCFA) and culturing in a selective broth medium. Final identification of Clostridium difficile was achieved by gas-chromatography and ApiZym. From the 207 consecutively obtained stool specimens, Clostridium difficile was isolated in 108 (52%) when pre-treated by heat-shock compared to only 26 (13%) when plated on modified CCFA and 23 (11%) when cultured in selective broth medium. Pre-selection significantly increases the isolation rate for Clostridium difficile and should be used in further epidemiological research. In the second part of our study, a retrospective review of subjects' records showed that the heat-shock method detected Clostridium difficile in all age groups at a higher rate than the other methods. In the third part of our study, we typed the 157 isolates of Clostridium difficile strains by protein patterns using SDS-PAGE, and 16 distinct groups were identified. In 19 cases different Clostridium difficile strains were found in the same subject by SDS-PAGE. Finally, the isolated strains were compared with strains from Brussels and Freiburg. Matching patterns were noted in only three cases.

Adolescent↗
PubMed2000

The effect of probiotics on Clostridium difficile diarrhea.

Clostridium difficile is the leading cause of nosocomially acquired intestinal infection in the United States, affecting virtually all cases of pseudomembranous colitis and up to 20% of cases of antibiotic-associated diarrhea. Even after receiving antibiotic treatment with either metronidazole or vancomycin, 20% of patients will have recurrent Clostridium difficile diarrhea. An innovative approach to the problem involves the introduction of competing, nonpathogenic (probiotic) organisms into the intestinal tract to restore microbial balance. The theoretical premise behind this approach is that the protective intestinal microflora is damaged by antibiotic treatment; the initial antibiotic exposure thus leaves the host susceptible to colonization and subsequent infection by Clostridium difficile. A so-called "second-hit" to the intestinal microflora occurs when the infected host is treated with flagyl or vancomycin, further destroying susceptible bacterial flora. Probiotic agents, such as Lactobacillus GG and Saccharomyces boulardii, have been studied for the treatment of Clostridium difficile. We are currently running a prospective, randomized, placebo-controlled trial of Lactobacillus GG in combination with standard antibiotics for the treatment of Clostridium difficile infection. Although it is too early to draw statistically significant conclusions, two patterns seem to be emerging: Lactobacillus GG is effective in reducing the 3-wk recurrence rate of Clostridium difficile, and patients feel better when taking Lactobacillus GG, as compared with the placebo, with early disappearance of abdominal cramps and diarrhea. In conclusion, the use of probiotics for the treatment of primary and recurrent Clostridium difficile diarrhea looks promising. Patients seem to have less recurrent Clostridium difficile diarrhea and early symptomatic improvement when using the probiotic Lactobacillus GG.

Aged↗
PubMed2005

Clostridium difficile causing acute renal failure: case presentation and review.

AIM: Clostridium difficile infection is primarily a nosocomial infection but asymptomatic carriers of Clostridium difficile can be found in up to 5% of the general population. Ampicillin, cephalosporins and clindamycin are the antibiotics that are most frequently associated with Clostridium difficile-associated diarrhea or colitis. Little is known about acute renal failure as a consequence of Clostridium difficile-associated diarrhea. METHODS: In this case report, we describe the course of Clostridium difficile-associated diarrhea in an 82-year-old patient developing acute renal failure. Stopping the offending agent and symptomatic therapy brought a rapid improvement of diarrhea and acute renal failure, full recovery was gained 18 d after admission. In a systematic review we looked for links between the two conditions. RESULTS: The link between Clostridium difficile-associated diarrhea and acute renal failure in our patient was most likely volume depletion. However, in experimental studies a direct influence of Clostridium difficile toxins on renal duct cells could be shown. CONCLUSION: Rapid diagnosis, nonspecific supportive treatment and specific antibiotic treatment, especially in the elderly, may lower excess mortality Clostridium difficile-associated diarrhea and renal failure being possible complications.

Acute Kidney Injury↗
PubMed2003

PCR detection of Clostridium chauvoei in pure cultures and in formalin-fixed, paraffin-embedded tissues.

The polymerase chain reaction (PCR) was used to amplify specific segments of the 16S ribosomal RNA gene of Clostridium chauvoei, a major pathogen of ruminants. Three sets of primers were used to produce amplicons of 159, 836 and 959 base pairs (bp), respectively. The PCR was evaluated by testing clinically important strains of Clostridium, including 21 strains of C. chauvoei, five strains each of Clostridium septicum and Clostridium perfringens and two strains each of Clostridium novyi, Clostridium histolyticum and Clostridium sordellii. Both purified DNA and biomass from pure cultures of each of these microorganisms were evaluated as templates in the PCR. In addition, extracts of formalin-fixed, paraffin-embedded tissues of eight sheep experimentally inoculated with C. chauvoei or C. septicum (four animals each) were also tested by the PCR using the three sets of primers. Purified DNA template of all C. chauvoei strains produced PCR amplicons of the expected size for all three primer pairs. However, when biomass from pure cultures of C. chauvoei or tissue extracts were used as templates, only the primer pair designed to produce the 159bp amplicon gave consistently positive results. No positive results were obtained with any primer pair when purified DNA or biomass from pure cultures of non-target clostridial species were used as templates. Therefore, the PCR primer sets appear to be very specific for identifying C. chauvoei in both cultures and tissues.

Animals↗
PubMed1979

Immunofluorescent study of the spore antigens of proteolytic strains of Clostridium botulinum.

By means of the spore fluorescent antibody technique 31 strains of Clostridium botulinum types A (18 strains), B (10 strains) and F (3 strains) were found to belong to the same homogeneous group irrespective of their toxigenic types. Some strains of this species also cross-reacted with certain strains of Clostridium sporogenes types I, II and III and Clostridium histolyticum type II. By spore antigenic analysis it was found that Clostridium parabotulinum contained two components designated L and M, the former describing species specificity; the latter was the cross-reacting component shared by some strains of Clostridium sporogenes and Clostridium histolyticum. Following this, a scheme showing the distribution of spore antigenic components among various species of Clostridium was given.

Antigens, Bacterial↗
PubMed2004

Clostridium infections associated with musculoskeletal-tissue allografts.

BACKGROUND: Allografts are commonly used in orthopedic reconstructive surgery. In 2001, approximately 875,000 musculoskeletal allografts were distributed by U.S. tissue banks. After the death from Clostridium sordellii sepsis of a 23-year-old man who had received a contaminated allograft from a tissue bank (Tissue Bank A), the Centers for Disease Control and Prevention initiated an investigation, including enhanced case finding, of the methods used for the recovery, processing, and testing of tissue. METHODS: A case of allograft-associated clostridium infection was defined as a culture-proven infection of a surgical site within one year after allograft implantation, from January 1998 to March 2002. We traced tissues to tissue banks that recovered and processed these tissues. We also estimated the rates of and risk ratios for clostridium infections for tissues processed by the implicated tissue bank and reviewed processing and testing methods used by various tissue banks. RESULTS: Fourteen patients were identified, all of whom had received allografts processed by Tissue Bank A. The rates of clostridium infection were 0.12 percent among patients who received sports-medicine tissues (i.e., tendons, femoral condyles, menisci) from Tissue Bank A and 0.36 percent among those who received femoral condyles in particular. The risk-ratio estimates for clostridium infections from tissues processed by Tissue Bank A, as compared with those from other tissue banks, were infinite (P<0.001) for musculoskeletal allografts, sports-medicine tissues, or tendons. Because Tissue Bank A cultured tissues only after treating them with a nonsporicidal antimicrobial solution, some test results were probably false negatives. Tissues from implicated donors were released despite the isolation of clostridium or bowel flora from other anatomical sites or reports of infections in other recipients. CONCLUSIONS: Clostridium infections were traced to allograft implantation. We provide interim recommendations to enhance tissue-transplantation safety. Tissue banks should validate processes and culture methods. Sterilization methods that do not adversely affect the functioning of transplanted tissue are needed to prevent allograft-related infections.

Adolescent↗
PubMed1976

Quantitative chemical analyses and antigenic properties of peptidoglycans from Clostridium botulinum and other clostridia.

The cell wall peptodoglycans were isolated from Clostridium botulinum and some other species of the genus Clostridium by hot formamide extraction and their quantitative chemical composition and antigenic properties were determined. The petidoglycan of C. botulinum type E was found to be a diaminopimelic acid (DAP)-containing type composed of glucosamine, muramic acid, glutamic acid, alanine and DAP in the molar ratio of 0.76:0.78:1.00:1.88:0.81. All other types of C. botulinum and Clostridium sporogenes also belonged to the same peptidoglycan type. The peptidoglycans of Clostridium bifermentans and Clostridium histoloyticum contained DAP but they differed from those of C. botulinum in the molar ratio of alanine to glutamic acid. The peptidoglycan of Clostridium perfringens was composed of glutamic acid, alanine, DAP and glycine in the molar ratio of 1.00:1.64:0.94:0.90. On the other hand, the peptidoglycan of Clostridium septicum was found to contain lysine instead of DAP and the molar ratio was 1.00:1.41:0.96 for glutamic acid, alanine and lysine. In spite of the difference in amino acid composition of peptidoglycans among the clostridia, the quantitative precipitin test demonstrated that antiserum against C. botulinum type E peptidoglycan cross-reacted with the peptidoglycans from other clostridia as well as various types of C. botulinum.

Alanine↗
PubMed1994

Distribution of Clostridium perfringens in different aquatic environments in Greece.

Clostridium perfringens and especially its spores, which are more tolerant to various physiochemical effects than the other fecal indicator bacteria, could serve as a useful indicator in ecosystems with stress factors. In order to determine the numbers of Clostridium perfringens in waters of rural North-West Greece, we utilised a new medium, lactose-sulfite (LS) broth, suggested for rapid enumeration and identification of Clostridium perfringens without the necessity for further confirmatory tests. Membrane filtration equipment was used on site. All samples were alternatively passed through two membrane filters, one (20-25 microns pore size) was used for retention of the abundant phytoplankton and the other (porosity 0.45 micron) for Clostridium perfringens. Membranes were placed into the first tube of 10-fold dilutions from 10(1) to 10(4) and incubated aerobically in a water-bath at 46 degrees C for 24 h. The numbers of Clostridium perfringens revealed fluctuations depending on the sampling site. It was significantly more common in the vicinity of industrial or domestic activities. LS broth not only allows the detection of small numbers of Clostridium perfringens but also permits rapid detection within 24 h. We propose the medium and the procedures described in this paper as a reliable technique for estimation of Clostridium perfringens as indicator of fecal contamination in waters.

Clostridium perfringens↗
PubMed2000

Phylogenetic characterization of several para- and meta-PCB dechlorinating Clostridium species: 16s rDNA sequence analyses.

The genus Clostridium has more than 127 species, grouped according to their morphology and functions. Nine Clostridium species were identified based on their ability to dechlorinate meta- and para-PCB (polychlorinated biphenyl) contaminated sediments. The phylogenetic relatedness of these PCB-degrading Clostridium species was studied using ribosomal RNA genes. The diversity of small-subunit rRNA genes associated with the domain bacteria was examined using defined operational taxonomic units (OTUs) in samples from PCB contaminated sediments from Lake Medinah, New York. The RFLP (restriction fragment length polymorphism) of the OTVs was measured. OTUs B (105 clones), A (33 clones) and C (45 clones) accounted for 75% of all the 16S rDNA clones expressing anaerobic para- and meta-PCB dechlorinating activity. In this report we describe complete 16S rDNA sequences of OTU-A and OTU-B, and partial rDNA sequences of OTUs C-J. The OTU-B and OTU-I form a phylogenetically related cluster, closely affiliated with Clostridium hydroxybenzoicum strains. OTUs A, C, D, G, H and J also belong to the genus Clostridium, but they represent separate species. OTU-E, a close affiliate to Bacteroides forsynthus, is a meta-PCB dechlorinator. The Cl. hydroxybenzoicum strains (OTU-B) are primarily para-PCB dechlorinators and are the most common. Some less prevalent OTUs (- E, -G, -H and -I), are also mostly para-PCB dechlorinators. Other Clostridium species such as Cl. beijerinckii (OTU-A), Cl. intestinalis (OTU-D) and Cl. thermolacticum (OTU-J) are primarily meta-PCB dechlorinators. Cl. paraputrificum (OTU-C) and Cl. cellulosi (OTU-F), were less prevalent in the total consortium, but they could dechlorinate both para- and meta-PCB. Although a few less prevalent Clostridium species can degrade both para- and meta-PCBs, this study confirms that para- and meta-PCB dechlorinating species are generally phylogenetically different.

Base Sequence↗
PubMed1993

Assignment of the agent of Tyzzer's disease to Clostridium piliforme comb. nov. on the basis of 16S rRNA sequence analysis.

The small-subunit rRNA (16S rRNA) sequence of Tyzzer's bacillus (also known as "Bacillus piliformis") was elucidated by using the polymerase chain reaction followed by reverse transcriptase sequencing. By using maximum-likelihood analysis, a phylogenetic tree was constructed from this and other 16S rRNA sequences available from the first release of the Ribosomal Database Project (G. J. Olsen, R. Overbeek, N. Larsen, T. L. Marsh, M. J. McCaughey, M. A. Maciukenas, W.-M. Kuan, T. J. Macke, Y. Xing, and C. R. Woese, Nucleic Acids Res. 20:2199-2200, 1992). Tyzzer's bacillus grouped with a specific set of anaerobic bacteria, most of which are Clostridium spp. The closest identified relatives are Clostridium coccoides, Clostridium oroticum, Clostridium clostridiiforme, Clostridium symbiosum, and Streptococcus hansenii. Clostridium amino-valericum and "Acetitomaculum ruminis" are also solidly allied with this ensemble. We propose that Tyzzer's bacillus be reclassified as Clostridium piliforme on the basis of its 16S rRNA sequence.

3T3 Cells↗
PubMed2003

Clostridium colicanis sp. nov., from canine faeces.

Morphological, biochemical and molecular genetic studies were performed on an unknown, anaerobic, rod-shaped organism isolated from faeces of a canine. The organism was tentatively identified as a member of the genus Clostridium based on its cellular morphology and ability to form endospores but, biochemically, it did not appear to correspond to any recognized species of this genus. Comparative 16S rRNA gene sequence analysis showed that the bacterium represents a previously unrecognized subline within Clostridium rRNA group I (Clostridium sensu stricto), which includes Clostridium butyricum, the type species of the genus. The nearest phylogenetic relatives of the unknown bacterium corresponded to Clostridium absonum, Clostridium baratii, Eubacterium budayi, Eubacterium moniliforme, Eubacterium multiforme and Eubacterium nitritogenes, but 16S rRNA sequence divergence values of > 3% demonstrated that it represents a novel species. Based on the findings presented, a novel species, Clostridium colicanis sp. nov., is described, with the type strain 3WC2T (=CCUG 44556T =DSM 13634T).

Animals↗
PubMed2006

The possible significance of Clostridium spp. in blood cultures.

The significance of Clostridium spp. in blood cultures was evaluated by two methods. In the first part of the study, a group of 80 patients with Clostridium spp. bacteraemia was compared with a group of 100 patients with Bacillus spp. in blood cultures, making the assumption that Bacillus almost invariably represents contamination (pseudobacteraemia). Significant differences were found between the two groups, suggesting that growth of Clostridium did not represent pseudobacteraemia. Patients with Clostridium bacteraemia were older, had a higher frequency of gastrointestinal disease (especially colorectal tumours), were associated more frequently with polymicrobial bacteraemia, and had a higher mortality rate. In the second part of the study, each of the 80 cases of Clostridium bacteraemia was evaluated individually for clinical relevance by an infectious diseases expert. In two-thirds of the cases, isolates of Clostridium from blood were considered to be of clinical relevance, whereas in one-third of cases, the clinical significance of this finding was doubtful. It was concluded that growth of Clostridium spp. in blood cultures, even in the absence of one of the histotoxic syndromes, is often of clinical significance, and that such findings should be properly evaluated and not ignored.

Aged↗
PubMed1999

Phylogenetic basis for a taxonomic dissection of the genus Clostridium.

The 16S rDNA-based phylogenetic analysis of the genus Clostridium has been completed by determination of the phylogenetic position of the type strains of 15 species and two non-validated species. These strains are members of phylogenetic clusters I, III, IV, V, IX, XIVa and XVIII as defined previously by Collins et al. [Int. J. Syst. Bacteriol. 44 (1994) 812-826]. Members of the genus Clostridium span a large evolutionary distance and the genus is not a phylogenetically coherent taxon but is intermixed with members of different genera, exhibiting a combination of Clostridium- and non-Clostridium-type properties. Anaerobacter polyendosporus, Syntrophococcus sucromutans and Acetivibrio multivorans also cluster within the radiation of Clostridium species. Although several taxa have been described for former Clostridium species with distinct phenotypic properties, the majority of Clostridium species, which are not members of the core cluster I, can at present not be reclassified as long as taxon-specific, phenotypic properties are not available.

Base Sequence↗
PubMed2004

Identification, detection, and spatial resolution of Clostridium populations responsible for cellulose degradation in a methanogenic landfill leachate bioreactor.

An anaerobic landfill leachate bioreactor was operated with crystalline cellulose and sterile landfill leachate until a steady state was reached. Cellulose hydrolysis, acidogenesis, and methanogenesis were measured. Microorganisms attached to the cellulose surfaces were hypothesized to be the cellulose hydrolyzers. 16S rRNA gene clone libraries were prepared from this attached fraction and also from the mixed fraction (biomass associated with cellulose particles and in the planktonic phase). Both clone libraries were dominated by Firmicutes phylum sequences (100% of the attached library and 90% of the mixed library), and the majority fell into one of five lineages of the clostridia. Clone group 1 (most closely related to Clostridium stercorarium), clone group 2 (most closely related to Clostridium thermocellum), and clone group 5 (most closely related to Bacteroides cellulosolvens) comprised sequences in Clostridium group III. Clone group 3 sequences were in Clostridium group XIVa (most closely related to Clostridium sp. strain XB90). Clone group 4 sequences were affiliated with a deeply branching clostridial lineage peripherally associated with Clostridium group VI. This monophyletic group comprises a new Clostridium cluster, designated cluster VIa. Specific fluorescence in situ hybridization (FISH) probes for the five groups were designed and synthesized, and it was demonstrated in FISH experiments that bacteria targeted by the probes for clone groups 1, 2, 4, and 5 were very abundant on the surfaces of the cellulose particles and likely the key cellulolytic microorganisms in the landfill bioreactor. The FISH probe for clone group 3 targeted cells in the planktonic phase, and these organisms were hypothesized to be glucose fermenters.

Base Sequence↗
PubMed1990

Enumeration and confirmation of Clostridium tyrobutyricum in silages using neutral red, D-cycloserine, and lactate dehydrogenase activity.

Spores of clostridia in big bale silages, manure, and dairy products were enumerated and distinguished from other spore formers by using Reinforced Clostridium Agar containing .005% neutral red. Spores of Clostridium tyrobutyricum predominated, but spores of Clostridium butyricum, Clostridium sporogenes, Clostridium bifermentans, Clostridium putrificum, and Clostridium sphenoides occurred to a lesser extent. In samples with high bacterial spore counts, growth of Bacillus spp., but not C. tyrobutyricum, was retarded by the addition of 200 ppm D-cycloserine. Clostridia isolated from silages and milk products were identified and tested on lactate dehydrogenase activity. Of 275 investigated strains, only strains identified as C. tyrobutyricum tested positively. Only 65% of the tested strains of C. tyrobutyricum grew in the confirmatory substrate containing minerals, lactic acid, and acetic acid. Tyrobutyricum Broth was not selective for C. tyrobutyricum, since C. butyricum and C. sporogenes also grew in this medium.

Animal Feed↗
PubMed1992

Clostridium difficile infection--a poor prognostic sign in uremic patients?

Uremia has been reported as a risk factor for the occurrence of infection with Clostridium difficile. During the two-year period 1984-86, 110 episodes of Clostridium difficile infection were encountered in 70 patients on a nephrology ward. Sixty-two patients had chronic renal failure and eight had acute renal failure. Sixty-seven of the patients were uremic and were treated with hemodialysis (n = 35), CAPD (n = 21), intermittent peritoneal dialysis (n = 6) or conservatively with a low protein diet (n = 5). Most of the patients were female (n = 41) and elderly (64 +/- 2 years). Malnutrition was common as indicated by low serum albumin concentrations (26 +/- 1 g/l) prior to the Clostridium difficile infection. Clostridium difficile infection was confirmed by stool culture and/or cytotoxin assay. Asymptomatic infections were found in eight patients. The highest relative risks of subsequent Clostridium difficile infection were calculated for patients treated with cephalosporins and isoxazolyl penicillins. All patients were treated with vancomycin, which often resulted in a dramatic improvement. One to six relapses of Clostridium difficile infection were observed in 22 of the patients. Sixty of the original 70 patients died during the five-year follow-up period. Thirty-four patients died during the first year of follow-up. Seven patients were transplanted, two are still on CAPD treatment and one has only moderate chronic renal failure (serum creatinine 200 mumol/l). Elderly debilitated uremic patients are especially susceptible to infection with Clostridium difficile which may be a poor prognostic sign in chronic renal failure.(ABSTRACT TRUNCATED AT 250 WORDS)

Clostridioides difficile↗
PubMed2006

Intravenous immunoglobulin for the treatment of severe, refractory, and recurrent Clostridium difficile diarrhea.

PURPOSE: Clostridium difficile diarrhea is common in elderly patients and leads to prolonged hospitalization. Patients with severe or recurrent Clostridium difficile diarrhea have poor antitoxin antibody responses. Intravenous immunoglobulin has been advocated in these patients. This study was designed to assess the response of patients with refractory, recurrent, or severe Clostridium difficile diarrhea to intravenous immunoglobulin. METHODS: Retrospective review (November 2003-January 2005) of 14 patients with severe, refractory, recurrent Clostridium difficile diarrhea treated with intravenous immunoglobulin (Flebogamma, 150-400 mg/kg) from 264 Clostridium difficile toxin-positive patients. RESULTS: Median age was 79 (range, 54-91) years. Median length of symptoms before intravenous immunoglobulin was 29 (range, 3-90) days. Patients received a median of three (range, 1-5) courses of vancomycin or metronidazole before intravenous immunoglobulin. All had hypoalbuminemia (median, 22 g/l; range, 18-33) and raised C-reactive protein (median, 47 mg/l; range, 25-255) at time of infusion. The median white cell count was 15.3 x 10(9)/liters (range, 4-24). Eight patients had evidence of pancolitis on abdominal imaging, suggesting severe Clostridium difficile diarrhea. All patients tolerated intravenous immunoglobulin without side effects. Nine (64 percent) responded with bowels normalizing in a median of ten (range, 2-26) days; one patient received two doses. One patient had a partial response from two doses but died two months later after a recurrence. The other four patients died of other causes within three weeks of infusion. CONCLUSIONS: Intravenous immunoglobulin may be effective for severe, refractory, or recurrent Clostridium difficile diarrhea after failed conventional treatment.

Aged↗
PubMed2000

Clostridium difficile infection in allogeneic stem cell transplant recipients is associated with severe graft-versus-host disease and non-relapse mortality.

We retrospectively evaluated 75 allogeneic stem cell transplant recipients to ascertain the incidence, risk factors and outcome of infection with Clostridium difficile. Ten patients (13%) had Clostridium difficile infection at a median of 38 days (range day -6 to day +72) following the transplant. There was no difference in the duration or severity of diarrhoea in patients with Clostridium difficile infection compared to the uninfected patients and no relationship to the prior antibiotic or chemotherapy usage, age, gender, underlying disease, donor type, CMV serostatus, total body irradiation or time to engraftment. The incidence of viral infections was increased in patients infected with Clostridium difficile (7/10 vs 15/65, P = 0.005, odds ratio 7.7), but the strongest association was with GVHD >grade 2 (5/10 vs 6/65 uninfected patients, P = 0.004, odds ratio 9.8). Patients infected with Clostridium difficile also suffered a higher non-relapse mortality with 7/10 patients succumbing to either GVHD or infections, compared to 19/65 patients in the uninfected group (P = 0.02, odds ratio 5.6). Thus Clostridium difficile infections in our study had a strong association with GVHD and increased non-relapse mortality. It is possible that Clostridium difficile toxin might predispose to increased severity of GVHD leading to an adverse outcome.

Adolescent↗