Isolation of Clostridium difficile from the Feces and the Antibody in Sera of Young and Elderly Adults

Abstract

Attempts were made to isolate Clostridium difJicile 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. difJicile 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. difJicile strains isolated produced cytotoxin which was neutralized by Clostridium sordellii antitoxin in vitro; in both young and elderly adults approximately 30% of the C. difJicile isolates were nontoxigenic. The mean concentration of C. difJicile in feces was 10 4 • 1/g in young adults and 104.6/g in elderly adults, with a range of 10 2 • 0 to 10 6 • 9/g. Antibody against C. difJicile toxin was found in most of the sera obtained from young adults carrying toxigenic C. difJicile, but not in sera of elderly adults, no matter how abundant was toxigenic C. difJicile in the feces.Toxigenic strains of Clostridium dijfu;ile have been shown to be a major cause of antibiotic-associated pseudomembranous colitis (PMC) of man (1,3,7,8,13). Feces from the vast majority of PMC patients contain a cytopathic toxin which is neutralized by Clostridium sordellii antitoxin, and toxigenic C. difficile in high concentration. Toxigenic strains of this organism have been isolated also at a high rate from feces of healthy neonates and infants (II, 15, 18) but rarely from those of healthy adults (6, 15). Kobayashi et al (14) isolated C. difficile at a high rate from the feces of healthy adults by using a selective medium devised by George et al (5), although there were fewer than 100 of the organisms per gram in most specimens. Recently, Willey and Bartlett (19) also developed a selective medium for the isolation of C. dijfu;ile, but failed to isolate the organism from the feces of healthy adults.Considering that antibiotic-associated PMC or diarrhea occurs more frequently in elderly adults (2, 10), we attempted to determine the frequency of isolation of C. difficile from fecal specimens, the toxigenicity of the isolates, and the presence of antibody against C. dijfu;ile toxin in the sera of young and elderly adults.

Effect of Arginine on Toxin Production by Clostridium difficile in Defined Medium

“…Twenty toxigenic C. difficile strains isolated in this laboratory from healthy adults or patients with antibiotic-associated diarrhea (14) were used.…”

A defined growth medium for Clostridium difficile

“…C. djicile strains were cultivated on modified cycloserine-cefoxitin-fructose-agar (CCFA) plates (Nakamura et al, 1981) anaerobically for 48 h. Several colonies of each strain were transferred to a medium for subculture (see Tables 2-4) and incubated at 37 "C for 10 h. The culture was then diluted 1000-fold in pre-reduced 0.85% (w/v) NaCl and 0.1 ml of the diluted culture was inoculated in duplicate into test media. The cultures were incubated at 37 "C. Inoculation, …”

Agglutination, Toxigenicity and Sorbitol Fermentation of Clostridium difficile

“…9689, 17857, and 17859 from the American Type Culture Collection (ATCC) , Rockville, Md., U.S.A.; 35 toxigenic and 15 nontoxigenic strains from healthy adults; five toxigenic and eight nontoxigenic strains from patients receiving antibiotics without gastrointestinal complications; eight toxigenic strains from antibiotic-associated diarrhea patients, and five toxigenic strains from antibiotic-associated PMC patients. Most of these strains were previously described (12,13 Preparation of antigen for the agglutination test. Bacterial cell antigen was prepared from a 7-hr culture of C. difJicile strain grown in a medium containing 3% (wjv) proteose peptone no.…”

“…Feces from the vast majority of PMC patients contain a cytopathic toxin which is neutralized by Clostridium sordellii antitoxin, and toxigenic C. difficile at high concentrations. The authors (12) previously reported that a fairly high population of young and elderly adults of Japanese harbored C. difficile in their colonic flora without any deleterious effect and that the antibody against C. difficile toxin was present in most young adults but not in elderly adults. With respect to the pathogenesis of antibiotic-associated diarrhea or PMC, it is claimed that in the presence of antimicrobial agents, C. difficile might selectively grow and produce a cytopathic toxin causing diarrhea or PMC (1,4).…”