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PubMed1991

[Response of the IRD intestinal epithelial cell line to Clostridium difficile toxins A and B in rats. Effect of Saccharomyces boulardii].

In vivo, Clostridium difficile acts by releasing 2 toxins: toxin A, an enterotoxin, and toxin B, a cytotoxin. This study was performed to determine: a) whether the rat epithelial intestinal cell line IRD 98 responds to Clostridium difficile toxin A and B; b) whether the yeast Saccharomyces boulardii has an effect on this model. Evaluation of 3H-thymidine incorporation into IRD 98 cells exposed to toxin B revealed that DNA synthesis was inhibited for low concentrations (10 ng/ml). For higher concentrations, DNA synthesis was not modified. Evaluation of 14C-leucine incorporation into IRD 98 cells exposed to toxin B revealed that this toxin affected protein synthesis. Whereas cholera toxin stimulates adenylate cyclase in IRD 98 cells, cAMP levels in cells exposed to various quantities of toxin A was similar to that in control cells. As opposed to cholera toxin, which does not affect the cytoskeletal structure of IRD 98 cells, 7 micrograms/ml of toxin A and even smaller amounts of toxin B (1 ng/ml) were found to cause structural alterations by immunofluorescence studies. Prior exposition of cells to Saccharomyces boulardii reduced or prevented the rounding of cells in the presence of these toxins. IRD 98 cells can thus be considered a good model for in vitro investigation of the effects of Clostridium difficile toxins A and B, and findings suggest that Saccharomyces boulardii has a protective effect against the action of these toxins.

Animals↗

PubMed1990

Prevalence of Clostridium difficile on a mixed-function ward for the elderly.

We studied the prevalence of Clostridium difficile over a period of 2 months on a mixed-function geriatric ward. Seven (14%) of the 49 patients were long-stay but the remainder were in hospital for acute illness or required a short period of active rehabilitation. Although 69% patients had recently received antibiotics, from only two (4%) was C. difficile isolated from their faeces. Our results show that C. difficile is not endemic in patients on all geriatric wards as has been previously suggested.

Aged↗

PubMed2005

A new periparturient disease in Eastern Europe, Clostridium difficile causes postparturient sow losses.

Postparturient sow losses caused by Clostridium difficile have not been reported in the veterinary literature. Recently in Croatia, in a large outdoor production unit with suboptimal environmental conditions, a sudden increase in postparturient sow mortality was diagnosed. After postpartal application of enrofloxacine to postparturient mastitis metritis agalactia (MMA) suffering sows, diarrhea, respiratory distress, and mortality of these sows were recorded. While 13% of MMA suffering and treated sows died, only 0.4% of the non-treated (no MMA suffering) sows died postpartum. Gross pathology revealed mesocolonic edema, hydrothorax, and ascites. Microscopic examination showed scattered foci of suppuration in the colonic lamina propria and accumulation of neutrophils and fibrin on colonic mucosa. Anaerobic cultures of the colon yielded heavy growth of C. difficile. Enzyme immunoassay revealed C. difficile toxins A and B. C. difficile infections of postparturient MMA suffering sows may be associated with environmental stress, the application of antibiotics, or both. C. difficile infections are an impending danger in Eastern Europe and does not only raise animal welfare issues, but seriously inflict the economical well being of outdoor production units.

Animals↗

PubMed1999

Low prevalence of nosocomial Clostridium difficile transmission, as determined by comparison of arbitrarily primed PCR and epidemiological data.

An increased prevalence of patients with C. difficile-associated diarrhoea in a hospital setting suggested the possible existence of an endemic occurrence. A study was therefore designed to determine clonal relatedness among 173 isolates of C. difficile, collected consecutively during 1995 from 147 patients (89 inpatients and 58 outpatients) and to estimate the probability of nosocomial transmission. Arbitrarily primed PCR (AP-PCR) with three different primers, AP1, AP2 and CLD1, was used for fingerprinting and identified 21, 92 and 70 types, respectively. Overall DNA analysis of the combined AP-PCR data yielded 140 types, of which 130 were unique, whereas 10 types occurred repeatedly in 36 isolates from 33 patients; seven isolates were non-typeable by one of the primers. Epidemiological data confirmed that in eight of the 33 patients there was a high probability of nosocomial transmission. Despite a high prevalence of C. difficile among hospitalized patients, a low frequency of nosocomial transmission was suggested by high resolution molecular typing of bacterial isolates in conjunction with traditional epidemiological methods.

Adult↗

PubMed2002

Rarity of toxigenic Clostridium difficile infections after hematopoietic stem cell transplantation: implications for symptomatic management of diarrhea.

Diarrhea is a common complication of high-dose chemotherapy and hematopoietic stem cell transplantation (HSCT). The frequent and prolonged use of multiple antibiotics in this setting can predispose to infection with toxigenic Clostridium difficile and the development of pseudomembranous colitis. Anti-motility agents are usually not administered in this setting until C. difficile infection has been excluded. The objective of this study was to determine the incidence of C. difficile toxin (CDT) positivity at the time of initial diarrhea in HSCT recipients, and to see if the practice of ensuring negative CDT assays prior to initiating symptomatic management of diarrhea needs modification. One hundred and nineteen patients with malignant diseases undergoing autologous or allogeneic HSCT were studied to determine the incidence of diarrhea and CDT positivity with initial diarrhea. One hundred and nine (91%) had diarrhea. Of these, only seven (6%) were CDT+ at the time of initial diarrhea. The median interval between onset of diarrhea and starting symptomatic anti-diarrheal therapy was 1 day. There were no significant differences between the patients with CDT+ diarrhea and the others in terms of timing or severity of diarrhea, number or duration of antibiotic usage, or leukocyte count. The infection resolved in all patients with metronidazole therapy. Our data suggest that the incidence of CDT+ diarrhea is low in HSCT recipients. Concern about C. difficile infection should not delay symptomatic therapy of initial diarrhea in HSCT recipients.

Adolescent↗

PubMed2001

Characteristics of Clostridium difficile strains isolated from asymptomatic individuals and from diarrheal patients.

OBJECTIVES: To characterise genotypes of Clostridium difficile strains isolated from asymptomatic individuals and patients with diarrhea. METHODS: Fecal specimens from 235 asymptomatic infants <12 months, 76 asymptomatic children 1-11 years and 132 adult patients with antibiotic-associated and non-antibiotic-associated diarrhea obtained from Siriraj Hospital, Bangkok from October 1998 to April 1999 were examined for C. difficile by cycloserine-cefoxitin-fructose agar culture. The presence of the C. difficile toxin A gene was determined by specific PCR with the use of primers 5-(CCC AAT AGA AGA TTC AAT ATT AAG CTT)-3 and 5-(GGA AGA AAA GAA CTT CTG GCT CAC TCA GGT)-3. All C. difficile isolates were subsequently genotyped by pulsed-field gel electrophoresis (PFGE). RESULTS: The C. difficile strains were found in 28 (11.9%) asymptomatic infants, 16 (21.1%) asymptomatic children and 33 (25%) adult patients. In total, 14 PFGE types and eight subtypes designated as types A, B, C, D, E, F, G, H, I, J, K, L, M and N, and A1, A2, A3, A4, B1, B2, B3 and E1, respectively, were identified. Only two isolates from infants and 18 isolates from adult patients were toxin A gene positive by PCR. Both isolates of toxigenic C. difficile were from infants in the same ward and were PFGE type B. PFGE type A was the predominant type among all toxigenic isolates (12 of 18 isolates) from adult patients. The other PFGE types of toxigenic C. difficile found in adult patients were: type A1, one isolate; type B, four isolates; and type C, one isolate. Types B2 and D were identified in 38.5% and 46.2%, respectively, of the toxin A gene-negative isolates of C. difficile from infants. CONCLUSIONS: These results revealed the occurrence of three distinct clusters from different wards in Siriraj Hospital. The toxigenic C. difficile of PFGE type A and related subtypes was a predominant infective clone in adult patients, whereas non-toxigenic C. difficile types B2 and D were encountered in asymptomatic infants. This information can be useful in epidemiologic investigations.

Adult↗

PubMed1997

Predicting Clostridium difficile stool cytotoxin results in hospitalized patients with diarrhea.

OBJECTIVE: To validate a model for the prediction of Clostridium difficile cytotoxin assay results, and to identify a subgroup of patients with a very low likelihood of C. difficile-associated disease in whom the yield of routine cytotoxin testing is low. DESIGN: Prospective cohort study. Relevant clinical symptoms, signs, and antibiotic exposure were recorded before reporting of assay results. Each predictor was assigned a score based on regression coefficients, and patients were stratified according to their total score. SETTING: Two urban, tertiary care, university hospitals. PATIENTS: A total of 609 consecutive adult inpatients who received testing for C. difficile cytotoxin during a 3-month period in 1994. MEASUREMENTS AND MAIN RESULTS: The prevalence of positive cytotoxin assays was 8% in the validation set, compared with 14% in the derivation set. Defining patients without both prior antibiotic use and at least one symptom predictor (significant diarrhea or abdominal pain) as a low-risk subgroup, the misclassification rate was 2.8% (5/177) for assay results; of the five misclassified cases patients, only one was judged to have C. difficile-associated disease. Use of this rule to identify low-risk patients could have potentially averted 29% of all cytotoxin assays. CONCLUSIONS: Patients without a history of antibiotic use and either significant diarrhea or abdominal pain are unlikely to have positive C. difficile cytotoxin assays and may not require cytotoxin testing.

Adult↗

PubMed2000

Value of lysozyme agar incorporation and alkaline thioglycollate exposure for the environmental recovery of Clostridium difficile.

Clostridium difficile is an increasingly prevalent nosocomial pathogen. Environmental contamination by spores is believed to be a major factor propagating the spread of C. difficile. Various approaches including the use of bile salts have been described to enhance the recovery of C. difficile from clinical and environmental specimens. We found that lysozyme (5 mg/L) incorporated into a selective medium containing bile salts significantly increased the recovery of C. difficile from swabs of 197 environmental sites (11% versus 24% samples positive, P< 0.01). Furthermore, in a separate series of experiments additional use of cooked meat broth enrichment significantly enhanced the recovery of C. difficile (35% versus 45%, P = 0.009). Conversely, we found that pre-exposure to alkaline thioglycollate did not improve the yield of C. difficile. Lysozyme incorporation markedly increases the recovery of C. difficile from environmental samples probably by stimulation of spore germination. Our findings suggest that previous attempts to determine the level of environmental C. difficile contamination have markedly underestimated the true prevalence of this pathogen.

Agar↗

PubMed2001

Bacterial contamination of uniforms.

Microbiological sampling of nurses' uniforms was undertaken using a Casella slit sampler. Staphylococcus aureus, Clostridium difficile and vancomycin-resistant enterococci were detected on uniforms both before and after a span of duty. Recommendations for provision and changing of nurses' uniforms are made.

Clostridioides difficile↗

PubMed1998

Clostridium difficile infection in patients with reactive arthritis of undetermined etiology.

In this study Clostridium difficile infection, which has been reported to induce reactive arthritis (ReA), was investigated in patients with ReA of undetermined etiology. One hundred patients with acute arthritis were included to in the study. The diagnosis of arthritis and/or infectious agents that are capable of causing ReA were determined in 69 of them. The remaining 31 patients (study group) with ReA of undetermined etiology were further investigated for C. difficile Toxin A (CDTA). The control groups were consisted of hospitalized patients and outpatients who had no history of diarrhea, arthritis, and antibiotic use. CDTA positive patients (19.4% of the study group) were treated only with oral vancomycin and evaluated for the prognosis of diarrhea and/or arthritis. The results strongly suggested C. difficile infection can induce ReA, especially in patients with antibiotic-associated colitis.

Acute Disease↗

PubMed1995

Evaluation of gas-liquid chromatography (GLC) for rapid detection of Clostridium difficile in fecal specimens.

Clostridium difficile intestinal infection is a major nosocomial hazard in patients receiving antimicrobial therapy. Rationale for rapid diagnosis include lifesaving antimicrobial therapy in patients with severe colitis and early isolation measures for transmission control. We have therefore analysed the sensitivity, specificity and predictive value of GLC identification of isocaproic acid in diarrheic stools from adult hospitalized patients in comparison with selective fecal culture on Cycloserine Cefoxitin Fructose Agar. During the study period, the prevalence of positive culture for C. difficile was 38/595 fecal specimens (6.4%). Compared with culture, GLC had a sensitivity of 24/38 (63%) and a specificity of 524/557 (94%). The predictive value of a positive GLC was 24/57 (42%) and of a negative GLC was 524/538 (97%). Measurement of the isocaproic acid peak height did not allow determination of a cutt-off value improving the test accuracy. The sensitivity of detection of isocaproic acid in stools by GLC is too low to be used as screening test for C. difficile infection. However, in a low prevalence population, a positive GLC test increased the pre-test probability of infection sevenfold.

Adult↗

PubMed2006

Diarrhea etiology in a Children's Hospital Emergency Department: a prospective cohort study.

BACKGROUND: We evaluated the frequency of recovery of pathogens from children with diarrhea who presented to a pediatric emergency department and characterized the associated illnesses, to develop guidelines for performing a bacterial enteric culture. METHODS: We conducted a prospective cohort study of all patients with diarrhea who presented to a large regional pediatric emergency department during the period from November 1998 through October 2001. A thorough microbiologic evaluation was performed on stool specimens, and the findings were correlated with case, physician, and laboratory data. RESULTS: A total of 1626 stool specimens were studied to detect diarrheagenic bacteria and, if there was a sufficient amount of stool, Clostridium difficile toxin (688 specimens), parasites (656 specimens), and viruses (417 specimens). One hundred seventy-six (47%) of 372 specimens that underwent complete testing yielded a bacterial pathogen (Shiga toxin-producing Escherichia coli, 39 specimens [of which 28 were serotype O157:H7]; Salmonella species, 39; Campylobacter species, 25; Shigella species, 14; and Yersinia enterocolitica, 2), a viral pathogen (rotavirus, 85 specimens; astrovirus, 27; adenovirus, 18; or rotavirus and astrovirus, 8), a diarrheagenic parasite (5 specimens); or C. difficile toxin (46 specimens). Samples from 2 patients yielded both bacterial and viral pathogens. A model to identify predictors of bacterial infection found that international travel, fever, and the passing of >10 stools in the prior 24 h were associated with the presence of a bacterial pathogen. Physician judgment regarding the need to perform a stool culture was almost as accurate as the model in predicting bacterial pathogens. CONCLUSIONS: Nearly one-half of the patients who presented to the emergency department with diarrhea had a definite or plausible pathogen in their stool specimens. We were unable to develop a model that was substantially better than physician judgment in identifying patients for whom bacterial culture would yield positive results. The unexpectedly high rate of C. difficile toxin warrants further examination.

Bacterial Toxins↗

PubMed1997

Colonic disease associated with a positive assay for Clostridium difficile toxin: a retrospective study.

In this retrospective review of colonic tissue from 21 patients with a positive stool assay for Clostridium difficile toxin, four groups of patients were identified by pathologic examination. Classic pseudomembranous colitis was identified in 38% of patients in colon biopsies, resections, and at postmortem examination. One third of patients had acute colitis without specific features on colon biopsies at the time of a positive toxin assay. Effects of C. difficile toxin in patients with idiopathic inflammatory bowel disease (10%) could not be pathologically separated from activity of the underlying disease. In 19% of patients, no acute or chronic colitis or pseudomembranous colitis was noted. This report reminds gastroenterologists that C. difficile infection is associated with a range of pathologic changes similar to the well known clinical spectrum of disease.

Adult↗

PubMed1993

Typing of Clostridium difficile strains by PCR-amplification of variable length 16S-23S rDNA spacer regions.

To develop a rapid and accurate method of typing large numbers of clinical isolates of Clostridium difficile, four regions of the rRNA operon [A, 15-1407 and B, 907-1407 (16S-16S); C, 1392-507 and D, 907-507 (16S-23S)] were enzymically amplified from 24 strains. When region A was hybridized to HindIII-digested genomic DNA isolated from C. difficile strains, all of the variable length restriction fragments hybridized. When region B was hybridized to HindIII-digested genomic DNA isolated from C. difficile strains, a set of variable length restriction fragments (Group II) hybridized predominantly. When region C was separated by agarose gel electrophoresis, a series of products ranging in size from approximately 800-1300 bp was obtained. When regions C and D were digested with HindIII, a constant region of 430 bp was found in both products and in all strains. From the above experiments it was concluded that the variable length Group II restriction fragments and the variable length region C amplification products were due to variable length 16S-23S spacer regions between alleles of the one strain. When region C amplification products were separated by denaturing PAGE, 16 variable length rRNA alleles (rrnA-P) were demonstrated from 24 C. difficile strains ranging in size from 852-1210 bp. After analysis with maximum parsimony, the 24 strains were divided into 14 ribotypes.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacterial Typing Techniques↗

PubMed2005

Detection and quantification of four species of the genus Clostridium in infant feces.

To determine the composition of Clostridium in the feces of infants approximately 30 days old, we have developed a detection and quantification method of Clostridium paraputrificum, Clostridium perfringens, Clostridium tertium, and Clostridium difficile by species-specific primers. C. perfringens and C. difficile were detected in four fecal samples from 22 infants (18.2%), whereas C. paraputrificum was detected in three samples (16.7%). C. tertium was detected in two samples (9.1%). Moreover, the occurrences of the four species in bottle-and mix-fed infants were relatively higher than in breast-fed infants (P< 0.05). Subsequently, positive samples detected by nested PCR (polymerase chain reaction) were subjected to realtime PCR. The results showed that the numbers of C. paraputrificum, C. perfringens, C. tertium, and C. difficile ranged from about 1x10(5) to 3x10(7) cells/g wet feces.

Bottle Feeding↗

PubMed1996

Anti-Clostridium difficile bovine immunoglobulin concentrate inhibits cytotoxicity and enterotoxicity of C. difficile toxins.

Clostridium difficile diarrhea and colitis result from the actions of bacterial exotoxins on the colonic mucosa. This study examined the ability of hyperimmune bovine colostral antibodies to neutralize the biological effects of these toxins. Anti-C. difficile bovine immunoglobulin concentrate was prepared from the colostral milk of Holstein cows previously immunized with C. difficile toxoids. The anti-C. difficile bovine immunoglobulin concentrate contained high levels of bovine immunoglobulin G specific for C. difficile toxins A and B, as evaluated by enzyme-linked immunosorbent assay. Anti-C. difficile bovine immunoglobulin concentrate neutralized the cytotoxic effects of purified toxin A and toxin B on cultured human fibroblasts, whereas control bovine immunoglobulin concentrate had little toxin-neutralizing activity. Anti-C. difficile bovine immunoglobulin concentrate also blocked the binding of toxin A to its enterocyte receptor and inhibited the enterotoxic effects of C. difficile toxins on the rat ileum, as measured by an increased rat ileal loop weight/length ratio (63% inhibition; P < 0.01), increased mannitol permeability (92% inhibition; P < 0.01), and histologic grading of enteritis (P < 0.01 versus nonimmune bovine immunoglobulin concentrate). Thus, anti-C. difficile bovine immunoglobulin concentrate neutralizes the cytotoxic effects of C. difficile toxins in vitro and inhibits their enterotoxic effects in vivo. This agent may be clinically useful in the prevention and treatment of C. difficile diarrhea and colitis.

Animals↗

PubMed1999

Antimicrobial activities of synthetic bismuth compounds against Clostridium difficile.

Clostridium difficile is a major nosocomial pathogen responsible for pseudomembranous colitis and many cases of antibiotic-associated diarrhea. Because of potential relapse of disease with current antimicrobial therapy protocols, there is a need for additional and/or alternative antimicrobial agents for the treatment of disease caused by C. difficile. We have synthesized a systematic series of 14 structurally simple bismuth compounds and assessed their biological activities against C. difficile and four other gastrointestinal species, including Helicobacter pylori. Here, we report on the activities of six compounds that exhibit antibacterial activities against C. difficile, and some of the compounds have MICs of less than 1 microgram/ml. Also tested, for comparison, were the activities of bismuth subcitrate and ranitidine bismuth citrate obtained from commercial sources. C. difficile and H. pylori were more sensitive both to the synthetic bismuth compounds and to the commercial products than were Escherichia coli, Pseudomonas aeruginosa, and Proteus mirabilis, and the last three species were markedly resistant to the commercial bismuth salts. Testing with human foreskin fibroblast cells revealed that some of the synthetic compounds were more cytotoxic than others. Killing curves for C. difficile treated with the more active compounds revealed rapid death, and electron microscopy showed that the bismuth of these compounds was rapidly incorporated by C. difficile. Energy dispersive spectroscopy X-ray microanalysis of C. difficile cells containing electron-dense material confirmed the presence of internalized bismuth. Internalized bismuth was not observed in C. difficile treated with synthetic bismuth compounds that lacked antimicrobial activity, which suggests that the uptake of the metal is required for killing activity. The nature of the carrier would seem to determine whether bismuth is transported into susceptible bacteria like C. difficile.

Anti-Bacterial Agents↗

PubMed2006

Analysis of proline reduction in the nosocomial pathogen Clostridium difficile.

Clostridium difficile, a proteolytic strict anaerobe, has emerged as a clinically significant nosocomial pathogen in recent years. Pathogenesis is due to the production of lethal toxins, A and B, members of the large clostridial cytotoxin family. Although it has been established that alterations in the amino acid content of the growth medium affect toxin production, the molecular mechanism for this observed effect is not yet known. Since there is a paucity of information on the amino acid fermentation pathways used by this pathogen, we investigated whether Stickland reactions might be at the heart of its bioenergetic pathways. Growth of C. difficile on Stickland pairs yielded large increases in cell density in a limiting basal medium, demonstrating that these reactions are tied to ATP production. Selenium supplementation was required for this increase in cell yield. Analysis of genome sequence data reveals genes encoding the protein components of two key selenoenzyme reductases, glycine reductase and d-proline reductase (PR). These selenoenzymes were expressed upon the addition of the corresponding Stickland acceptor (glycine, proline, or hydroxyproline). Purification of the selenoenzyme d-proline reductase revealed a mixed complex of PrdA and PrdB (SeCys-containing) proteins. PR utilized only d-proline but not l-hydroxyproline, even in the presence of an expressed and purified proline racemase. PR was found to be independent of divalent cations, and zinc was a potent inhibitor of PR. These results show that Stickland reactions are key to the growth of C. difficile and that the mechanism of PR may differ significantly from that of previously studied PR from nonpathogenic species.

Amino Acid Isomerases↗