Clostridium Sordellii Antitoxin Research Articles

Effect of filtrate containing Clostridium difficile toxin on rectal mucosa maintained in organ culture.

Rectal biopsies were maintained in organ culture over a 24-hour culture period, with good preservation of histological architecture. A filtrate containing Clostridium difficile toxin significantly inhibited the rise in epithelial alkaline phosphatase activity normally seen during culture. This effect was abolished by pre-incubation of the filtrate with Clostridium sordellii antitoxin, or heat inactivation. This effect is most probably due to a toxin of C. difficile. The method provides a new quantitative approach to the study of luminal toxins as possible pathogenic agents in idiopathic inflammatory diseases of the colon.

Treatment of antibiotic-associated pseudomembranous colitis.

The experience of this laboratory with the treatment of Clostridium-induced colitis in experimental animals and in patients was reviewed. Optimal results in hamsters were achieved with the antibiotics vancomycin, metronidazole, and tetracycline. Cholestyramine was less effective. The outcome for animals given corticosteroids and Clostridium sordellii antitoxin systemically was not different from that for untreated control animals. The second facet of the study was a retrospective review of therapy in 272 patients with C. difficile-induced diarrhea or colitis. No specific therapy was given to 56 patients who had mild symptoms or were improving at the time the toxin was detected. The therapy most frequently used was oral vancomycin, which was given to 189 patients, including 100 with confirmed pseudomembranous colitis. The response rate was 97%, but 46 patients (24%) relapsed when treatment was discontinued. Response to cholestyramine was favorable in 12 of 19 patients. The results with metronidazole and bacitracin were uniformly good, although the experience was limited.

Medical Management of Antimicrobial‐Associated Diarrhea and Colitis

Gastrointestinal complications, including diarrhea, may occur with virtually all antimicrobial agents. Such diarrhea may represent either a common, nonspecific adverse effect, or it may be one of the manifestations of antimicrobial-associated colitis (AAC), a potentially fatal complication. Clostridium difficile and a cytotoxin neutralized by Clostridium sordellii antitoxin has been isolated from the stools of nearly all patients with antibiotic-associated pseudomembranous colitis, many patients with AAC, and approximately 20% of those with antimicrobial-induced diarrhea. Demonstration that C. difficile is responsible for cytotoxin production has allowed for specific therapy for these disorders. General treatment measures include discontinuation of the causative antimicrobial agent(s), bowel rest, and supportive care with fluids, electrolytes and colloids, if necessary. Antiperistaltic agents and corticosteroids are not recommended. Various antimicrobials demonstrate potential efficacy in treating AAC in humans. Oral vancomycin is the most widely tested and is currently the treatment of choice. It achieves high concentrations in the feces and is very active against C. difficile in doses of 125-500 mg by mouth every six hours. Other potentially useful but inadequately tested antimicrobials include metronidazole (500 mg by mouth every eight hours) and bacitracin (25,000 units by mouth every six hours). Tetracycline has been employed with some success in nonspecific antibiotic-associated diarrhea, although it is as yet untested in humans with AAC and may induce diarrhea itself. Both miconazole and rifampin are highly effective against C. difficile in vitro but have not been evaluated in AAC. Anion-exchange resins bind the cytotoxin found in stools of patients with AAC. Cholestyramine has been used with variable response in oral doses of 4 g every six to eight hours. Since these resins may also bind vancomycin, resulting in lowered vancomycin concentrations in the stool, combination therapy should be used cautiously. With specific therapy directed against the toxin and aggressive supportive therapy, surgical intervention is rarely necessary. More recently, investigations have been directed at using bacterial preparations to suppress C. difficile by restoring the normal flora. The development of immunological agents (i.e., vaccines, toxoids, antitoxins) for the prevention or treatment of AAC would be a significant advance in therapy.

Isolation of Clostridium difficile from the feces and the antibody in sera of young and elderly adults.

Attempts were made to isolate Clostridium difficile from a total of 431 fecal specimens from 149 young and 213 elderly healthy adults, and 69 elderly adults with cerebrovascular disease but no gastrointestinal disease. C difficile was isolated from 49 specimens, and the frequency of isolation was 15.4% in healthy young adults, 7.0% in healthy elderly adults, and 15.9% in elderly adults with cerebrovascular disease. Thirty-four (about 70%) of the 49 C. difficile strains isolated produced cytotoxin which was neutralized by Clostridium sordellii antitoxin in vitro; in both young and elderly adults approximately 30% of the C. difficile isolates were nontoxigenic. The mean concentration of C. difficile in feces was 10(4.1)/g in young adults and 10(4.6)/g in elderly adults, with a range of 10(2.0) to 10(6.9)/g. Antibody against C. difficile toxin was found in most of the sera obtained from young adults carrying toxigenic C. difficile, but not in sera of elderly adults, no matter how abundant was toxigenic C. difficile in the feces.

Antibiotic-Associated Pseudomembranous Colitis in Children

Ten cases of antibiotic-associated pseudomembranous colitis in children are reviewed. The ages ranged from 4 years to 17 years; the most frequently implicated antimicrobial agents were penicillins in six children and clindamycin in two. Stool assays showed specimens from all ten patients yielded a cytopathic toxin which was neutralized by Clostridium sordellii antitoxin with titers ranging from 1:40 to 1:40,000. Bacterial cultures of nine specimens uniformly yielded Clostridium difficile with a median concentration of 10(5.4) organisms per gram of wet weight. All nine isolates of C difficile showed a vitro production of a cytopathic toxin which was similar to or identical with that which was detected in the original stool specimen. All ten patients recovered. Six were treated with oral vancomycin and showed a good therapeutic response; one patient, however, suffered two relapses when treatment was discontinued, requiring a total of three courses of oral vancomycin. Two patients received cholestyramine and responded well. These observations provide supportive evidence that C difficile is responsible for antibiotic-associated pseudomembranous colitis in children and document efficacy of the newer therapeutic modalities in this patient population as well.

Role of Clostridium difficile in a case of nonantibiotic-associated pseudomembranous colitis

A 57-yr-old woman with chronic diarrhea, mild azotemia, and red cell casts in her urine was found to have pseudomembranous colitis. She had not received antimicrobial agents for at least 2 yr. Membranoproliferative glomerulonephritis was found on kidney biopsy, and her renal function improved spontaneously. Pseudomembranous colitis was diagnosed by endoscopic biopsy. Her stool contained a cytopathic toxin that was neutralized by Clostridium sordellii antitoxin, and Clostridium difficile was cultured on selective media. This case indicates that C. difficile may be a cause of nonantibiotic-associated pseudomembranous colitis, as well as antibiotic-associated pseudomembranous colitis.

In vitro susceptibility of Clostridium difficile isolates from patients with antibiotic-associated diarrhea or colitis.

In vitro susceptibility tests were performed on 84 strains of Clostridium difficile to 11 antimicrobial agents. All isolates were from the stools of patients with antibiotic-associated diarrhea or colitis in which there was a cytopathic toxin that was neutralized by Clostridium sordellii antitoxin. Over 95% of the strains were susceptible to vancomycin, penicillin G, ampicillin, and metronidazole at concentrations of 4 microgram/ml. Susceptibility to clindamycin was variable; 60% of the strains were susceptible at 1 microgram/ml, and 9% were resistant at 128 microgram/ml. Studies of individual isolates showed that a major portion of the strains were relatively susceptible to the antimicrobial agent implicated in causing the disease.

Cecal toxin(s) from guinea pigs with clindamycin-associated colitis, neutralized by Clostridium sordellii antitoxin.

The cecal contents of guinea pigs with clindamycin-associated colitis contained a heat-labile toxin. This toxin was lethal for guinea pigs and mice, produced vascular permeability in the skin of rabbits, and was cytotoxic in tissue culture. The lethality in mice, vascular permeability in rabbit skin, and cytotoxicity in tissue culture monolayers were neutralized by Clostridium sordellii antitoxin.

Cultures for Clostridium difficile in stools containing a cytotoxin neutralized by Clostridium sordellii antitoxin.

Stools from patients with antibiotic-associated diarrhea or colitis were cultured to detect the presence of Clostridium difficile. All specimens contained a cytotoxin which was neutralized by Clostridium sordellii antitoxin. Initial testing employed several methods with comparative merits in recovering this organism. These included the use of nonselective media, antibiotic-incorporated media, alcohol shock, and paracresol-containing broth. Optimal results were achieved with primary plating of serial dilutions onto a selective agar containing cycloserine and cefoxitin. This technique was then employed in a large number of specimens. The overall results showed that C. difficile was recovered in specimens from 71 of 73 patients. All isolates of C. difficile produced a cytotoxin which was neutralized by C. sordellii antitoxin in vitro. These results verify the utility of this medium and support the concept that C. difficile accounts for the cytotoxin found in stools in nearly all cases.

Cytotoxicity Assay in Antibiotic-Associated Colitis

Stools were tested for the presence of a cytopathic toxin that is neutralized by gas gangrene antitoxin or Clostridium sordellii antitoxin. The test was positive in specimens from 51 (96070) of 53 patients with antibiotic-associated pseudomembranous colitis and five (2%) of 248 individuals without gastrointestinal complications of antimicrobial usage. Seven types of tissue cultures were tested, and all proved susceptible to this toxin. Those that appeared to be the most susceptible on the basis of titers of toxin in stools were primary human amnion cells, WI-38 cells, baby hamster kidney cells, and mouse fibroblasts. Storage of specimens at -70 C, 6 C, 28 C, and 37 C for five days before testing resulted in decrease in toxin titers that correlated with increasing temperatures. Studies of various C. sordellii antitoxin preparations indicated considerable variation in potency. Recommendations for performing the tissue culture assay for the toxin found in stools of patients with antibiotic-associated colitis are provided on the basis of these observations. Several recent reports indicate that stools from pa

Etiology of tetracycline-associated pseudomembranous colitis in hamsters.

Tetracyclines were implicated in the 1950s in induction of protracted diarrhea and pseudomembranous colitis. Because the pathogenetic mechanism of these illnesses has been questioned recently, we studied tetracycline in hamster models of antibiotic-associated colitis. Orogastric administration of tetracycline caused diarrhea and death, with evidence of hemorrhagic typhlitis. Filtrates of cecal contents were toxic when inoculated into normal hamsters and cell culture monolayers, and toxicity was neutralized with Clostridium sordellii antitoxin. Tetracycline-resistant C. difficile was cultured from stools of these hamsters, but Staphylococcus aureus was not isolated. The value of tetracycline for treatment or prevention of clindamycin-induced colitis in hamsters was also studied, and it was found that daily orogastric administration of tetracycline was poorly protective against clindamycin-induced colitis.

Antibiotic-Associated Pseudomembranous Colitis

C. difficile was originally described in 1935 but was of minimal clinical interest until recently, when studies showed it to be the cause of antibiotic-associated PMC. Intestinal complications caused by C. difficile appear almost exclusively in conjunction with exposure to antibiotics. Evidence that supports the etiologic role of this organism in antibiotic-associated PMC comes from both bacteriological studies and tissue culture assays. Tissue culture assays have been used to demonstrate that the stool supernate from nearly all patients with anatomically confirmed PMC contains a cytotoxin that is neutralized with Clostridium sordellii antitoxin. The cytotoxin has rarely been found in persons without gastro-intestinal complications related to antibiotic usage. Cultures of stool from patients with anti-biotic-associated PMC usually yield C. difficile, which is encountered only infrequently in the fecal flora of healthy persons. Strains of C. difficile have been shown to cause a similar disease in experimental animals administered antibiotics or intracecal injections of broth cultures of C. difficile. According to the antitoxin neutralization tests and biochemical purification studies done to date, C. difficile is the only organism that produces a cytotoxin similar or identical to the cytotoxin encountered in stools of patients with PMC. The toxin is a high-molecular-weight protein that appears to cause changes in the permeability of cells in tissue culture. However, the mechanism of its action on intestinal mucosa and the cause of its production with antibiotic exposure in clinical settings have not been well defined.

Partial Purification of a Toxin Found in Hamsters with Antibiotic-Associated Colitis

A toxin with cytotoxic and enterotoxic activities was isolated from cecal contents of hamsters receiving lincomycin. The toxin was partially purified by ultracentrifugation, ultrafiltration, (NH4)2SO4 precipitation, and gel filtration. Cytotoxic activity, assayed on monolayers of HeLa cells, was restricted to material that eluted in the molecular weight range of 107,000 +/- 6,000 daltons. Cytotoxicity of crude AAC toxin could be demonstrated at concentrations as low as 0.04 microgram/ml. The toxin was heat labile (55 degrees-60 degrees C for 0.5 hr) and sensitive to trypsinization, acidification at pH 3, or alkalinization at pH 9. Cytotoxic activity was inhibited by Clostridium sordellii antitoxin. Enterotoxic activity of the crude toxin and the cytotoxic fraction from gel filtration was demonstrated by fluid secretion in ligated rabbit ileal loops. Studies were done in vitro with cholestyramine resin, vancomycin, or gentamicin to determine if the toxin was bound or denatured by these drugs. It was demonstrated that cholestyramine bound the toxin, significantly reducing its cytotoxicity. Reversible binding of the cytotoxic material was demonstrated by salt gradient elution. Neither vancomycin nor gentamicin had any effect on the in vitro cytotoxic activity of the toxin.

Prevention of clindamycin-induced colitis in hamsters by Clostridium sordellii antitoxin

Toxins produced by Clostridium difficile have been implicated in the etiology of antibiotic-induced colitis. Clostridium difficile antitoxin is not available, but recent studies have shown that toxins present in the feces of patients with this disease are neutralized by Clostridium sordellii antitoxin. We found that C. sordellii antitoxin neutralized toxins produced in broth cultures of either C. sordellii or C. difficile and that passive immunization with C. sordellii antitoxin before challenge with clindamycin prevented colitis in hamsters. Significantly fewer antitoxin-treated animals than unimmunized controls developed diarrhea and died with hemorrhagic colitis. Administration of 300 U of antitoxin parenterally either on the day of challenge with clindamycin or 24 hr later provided significant protection (25% mortality vs. 100% mortality in controls, P < 0.01). None of eight animals given antitoxin (300 U) both on the day of challenge and 24 hr later died. Filtrates prepared from cecal contents of dead or killed hamsters were tested for toxicity by intraperitoneal injection into hamsters and by addition to monolayers of monkey kidney cells. Fecal filtrates from antitoxin-protected animals were not toxic in these assays, but filtrates from control animals were uniformly toxic. Passive immunization against clostridial toxins was protective against clindamycin-associated colitis in this model. This finding further substantiates the importance of these toxins in the pathogenesis of antibiotic-induced colitis.

Role of Clostridium difficile in antibiotic-associated pseudomembranous colitis

Tissue cultures were performed on stools from 189 patients to detect a cytopathic toxin which is neutralized by Clostridium sordellii antitoxin. Specimens satisfying these criteria were considered positive in the tissue culture assay. Stools from 26 of 27 patients with antibiotic-associated pseudomembranous colitis were positive and 16 of these specimens showed toxin titers of 10−3 dilutions or greater. The tissue culture assay was positive with specimens from 9 of 63 patients with antibiotic-associated diarrhea without documented pseudomembrane formation. Stools from patients with neonatal necrotizing enterocolitis, ulcerative colitis, and healthy controls were uniformly negative in this assay. Cultures were performed on stools from 38 patients with antibiotic-associated diarrhea or colitis to detect clostridia which produce a cytopathic toxin in vitro. Clostridium difficile was recovered from 6 of 8 specimens which were positive in the tissue culture assay and 5 of 30 which were negative in this assay. C. sordellii was recovered in a single specimen. One hundred and nine clostridia strains were tested in the tissue culture assay and C. difficile was the only species which produced a cytopathic toxin. All strains of this organism were positive in the tissue culture assay and, in each instance, cytotoxicity was neutralized by C. sordellii antitoxin. These results indicate that C. difficile is the major cause of antibiotic-associated pseudomembranous colitis and offer an explanation for previous studies showing that the cytotoxin of stools from these patients is neutralized by C. sordellii antitoxin.

Neutralization of Clostridium difficile toxin by Clostridium sordellii antitoxins.

Neutralization of Clostridium difficile toxin by Clostridium sordellii antitoxin was studied by cytotoxicity assay in tissue culture. The sources of toxin were stools from two patients with pseudomembranous colitis and a culture filtrate of C. difficile isolated from one of the patients. C. sordellii antitoxin was available either in monovalent form or as gas gangrene polyvalent antitoxin. The potency of antitoxins against C. difficile determined by cytotoxicity assay did not correlate with the established values reported for mouse protection tests against C. sordellii toxin. An equivalent zone of optimal neutralization was demonstrated for stool toxin, and a slightly different one for culture toxin. The rate of neutralization appeared to be instantaneous, either at 24 or at 37 degrees C. The efficacy of antitoxin in preventing cytotoxicity in cultured cells preexposed to toxin decreased rapidly with preexposure time. The union between toxin and antitoxin could be readily dissociated by simple dilution or by ammonium sulfate precipitation followed by dissociated by simple dilution or by ammonium sulfate precipitation followed by dilution. Continued incubation of toxin-antitoxin mixture did not increase the firmness of the union; on the contrary, more dissociation occurred. The unusual looseness of the toxin-antitoxin union is probably relatd to lack of serological specificity or affinity. Based on these observations, a practical diagnostic method for antibiotic-induced colitis is outlined.

Neutralization by Clostridium sordellii antitoxin of toxins implicated in clindamycin-induced cecitis in the hamster

A fecal toxin has previously been identified and implicated in the pathogenesis of clindamycin-induced cecitis in humans and hamsters. This report documents neutralization of the hamster toxin by Clostridium sordellii antitoxin and suggests its similarity to fecal toxins found in patients with antibiotic-associated colitis. Filtrates of broth cultures of C. sordellii and Clostridium difficile were also toxic for hamsters and cell cultures. The toxicity of filtrates from both organisms was neutralized by C. sordellii antitoxin.

PSEUDOMEMBRANOUS COLITIS: PRESENCE OF CLOSTRIDIAL TOXIN

A toxin was found in the fæces of nine out of nine patients with pseudomembranous colitis and two out of two with antibiotic-associated non-specific colitis. The toxin was neutralised by Clostridium sordellii antitoxin but not by a commercial mixture of C. welchii, œdematiens, and septicum antitoxins. The in-vitro and in-vivo properties of pseudomembranous-colitis toxin closely resemble those of the toxin of C. sordellii.

ANTIBIOTIC-INDUCED COLITIS IMPLICATION OF A TOXIN NEUTRALISED BY CLOSTRIDIUM SORDELLII ANTITOXIN

A toxin(s) has been demonstrated in the stools of two patients with antibiotic- associated colitis. This toxin(s) was heat-labile, wasrapidly lethal for hamsters, increased vascular permeability in rabbit skin, and was cytotoxic for cells in tissue-culture. It was neutralised by Clostridium sordellii antitoxin but not by antitoxins prepared against otherclostridia; Escherichia coli, and Vibrio cholerœ toxins. These characteristics were identical to those of a toxin implicated in the ætiology of antibiotic-induced colitis in the hamster. One patient improved rapidly after treatment with oral vancomycin, and at the same time the toxin disappeared from the stool.