Frequency-pulsed electron capture gas-liquid chromatographic analysis of metabolites produced by Clostridium difficile in broth enriched with amino acids.

Clostridium difficile strain CDC A-567 was cultured in Trypticase (BBL Microbiology Systems)-yeast-salt broth supplemented with 0.2% L-leucine, L-norleucine, L-isoleucine, L-tyrosine, or L-tryptophan. Four extractions were done on the spent medium, three at pH 2 and one at pH 10, using CHCL3 or ether. Derivatizations were done with trichloroethanol, heptafluorobutyric anhydride, and heptafluorobutyric anhydride-ethanol. All samples were analyzed with frequency-pulsed electron capture gas-liquid chromatography. A dedicated computer was used to assist in data analysis. C. difficile produced both short-chain and aromatic acids in Trypticase-yeast-salt broth; hydroxy acids were also detected. p-Cresol, indoleacetic acid, 4-methylthio-2-hydroxybutyric acid, and some unidentified alcohols were observed. The basic chloroform extraction contained cadaverine and putrescine. Leucine, norleucine, and isoleucine influenced the production of C5 and C6 acids and alcohols. L-Tyrosine underwent successive degradation to produce p-cresol and aromatic acids as final products. Tryptophan increased the production of indoleacetic, indolepropionic, and indolebutyric acids. Isocaproic acid was produced in relatively high concentrations regardless of medium substitution. The consistent production of iC6 under various substrate conditions indicates that the production of this compound might be consistent enough in vitro to form the basis of a rapid test for detection of C. difficile in stool specimens by frequency-pulsed electron capture gas-liquid chromatography.

Studies of stools from pseudomembranous colitis, rotaviral, and other diarrheal syndromes by frequency-pulsed electron capture gas-liquid chromatography.

Thirty-five patients with various diarrheal syndromes and 22 controls were studied. All stool samples were carefully cultured for Clostridium difficile, using selective isolation media. Cytotoxin assays with proper antitoxin neutralization were done in MRC-5 cells. The stool samples were extracted four times, three times at pH 2 and once at pH 10, using CHCl3 or ether. Derivatizations of extracts were done with trichloroethanol, heptafluorobutyric anhydride, and heptafluorobutyric anhydride-ethanol, and all derivatives were analyzed by frequency-pulsed electron capture gas-liquid chromatography (FPEC-GLC). A dedicated computer was used to assist in both qualitative and quantitative data analysis. Isocaproic acid (iC6) was always found in stool from which C. difficile was isolated and was absent in C. difficile-negative specimens. p-Cresol was found frequently in both persons with pseudomembranous colitis and controls. Tryptamine was found in stool containing C. bifermentans. The FPEC-GLC profiles of persons with acute diarrhea were very different from those of normal persons. Diarrhea associated with adenovirus and rotavirus, Klebsiella spp., and Escherichia spp. showed different FPEC-GLC patterns. Stools from well persons consistently contained full-scale peaks of pyruvic, acetic, propionic, isobutyric, butyric, isovaleric, and valeric acids. In rotavirus stools isobutyric, isovaleric, and valeric acids were reduced in quantity from those found in control stools, whereas propionic and butyric acids were increased.

Clostridium difficile. Colonization and toxin production in a cohort of patients with malignant hematologic disorders.

We examined 45 (80%) of 56 consecutive adult patients with malignant hematologic disorders who were hospitalized during a 15-week period at Emory University Hospital, Atlanta. Stool samples for Clostridium difficile culture and cytotoxin assay were obtained on admission and then weekly during each patient's hospitalization. On admission, four patients had detectable C difficile in their stool samples, which was associated with prior antimicrobial use but not with prior cancer chemotherapy. One of the four patients with positive stool samples also had toxin present in the stool sample and was the only one with diarrhea. Eight (36%) of 22 patients hospitalized for one or more weeks had C difficile isolated from at least one stool specimen. The positive cultures showed no clustering in time, and no risk factors were identified for colonization. Only seven of 15 culture-positive stool samples and three of seven toxin-positive samples were associated with diarrhea.

Norleucine-tyrosine broth for rapid identification of Clostridium difficile by gas-liquid chromatography.

A new medium, norleucine-tyrosine (NT) broth, was developed for rapid identification of Clostridium difficile on the basis of caproic acid and p-cresol production. The NT broth consists of 0.5% Trypticase (BBL Microbiology Systems, Cockeysville, Md.), 0.5% yeast extract (Difco Laboratories, Detroit), 0.2% L-norleucine and 0.2% L-tyrosine (wt/vol; final concentrations), and a mixture of salts. The procedure for demonstrating caproic acid and p-cresol production involves extracting NT broth cultures with ether or chloroform and analyzing the extracts with a gas-liquid chromatograph equipped with a thermal conductivity detector, as is customary for analysis of volatile fatty acids. A total of 120 strains of C. difficile from diverse geographic locations were tested by this procedure, and they all produced caproic acid and p-cresol in NT broth. No other Clostridium species or other microorganisms tested have been found to produce both products in NT broth.

Survey of the extrachromosomal gene pool of Clostridium difficile.

Pseudomembranous colitis, a severe diarrheal disease, has been linked to the administration of antibiotics and to two toxins produced by Clostridium difficile. Eighty-two strains of C. difficile isolated from humans and hamsters were assayed for the presence of plasmid DNA. Agarose gel electrophoresis of Sarkosyl-lysed cells indicated that 18% of the strains contained from one to four plasmids. The plasmid DNA in these strains ranged in molecular weight from 2.7 X 10(6) to 60 X 10(6). Strains with and without plasmids were examined for the cytopathogenic effect of the toxins on MRC-5 cells. No correlation was observed between plasmid content and cytopathogenic effect. The results of in vitro antibiotic susceptibility testing with plasmid-containing strains revealed that 33% of the strains tested exhibited growth with four or more of the antimicrobial agents used.

Antibiotic-associated pseudomembranous colitis: an epidemiologic investigation of a cluster of cases.

Ten cases of antibiotic-associated colitis (AAC) were identified at a hospital in Washington, D.C., from March 17 to May 9, 1979. No geographic clustering of cases was found, nor was an association with increased use of antibiotics demonstrated. Exposure to aminoglycosides, cephalosporins, and clindamycin was associated with AAC, as was a history of enemas in the seven days before the onset of illness (P=0.045). This association was strengthened when gastrointestinal procedures-defined as (1) three or more enemas per week, (2) the insertion of a nasogastric tube for two or more days, or (3) gastrointestinal surgery-were performed within seven days of the onset of illness (P=0.007). Clostridium difficile was not isolated from the hospital environments, nursing personnel, or family members of the patients. C. difficile was isolated from stool specimens of five (36%) of 14 patients who served as controls.

Analysis of short-chain acids from bacteria by gas-liquid chromatography with a fused-silica capillary column.

The use of a flexible, fused-silica capillary column for gas-liquid chromatographic analysis of short-chain acids from bacteria is illustrated with a standard acid mixture and with a derivatized extract of culture medium from Clostridium difficile.

Diet as a coadjuvant for development of antibiotic-associated diarrhea in hamsters (Mesocricetus auratus).

During a study of the effects of nutrition on experimental tumors in hamsters, fatal diarrhea developed. To determine the role of diet in this condition and the relationship between diet and antibiotics, two diets were used, ground commercial diet and a purified diet. Two antibiotics were used, neomycin sulfate and vancomycin. Diarrhea was evident soon after the animals were given the combination of purified diet and neomycin sulfate. Vancomycin initially acted as a suppressor of diarrhea, but hamsters fed the purified diet developed diarrhea after the drug was discontinued. None of the animals fed ground commercial diet treated with neomycin sulfate developed diarrhea. Clostridium difficile was isolated from most of the sick animals; all of the isolates of Clostridium difficile were toxigenic except one. No significant differences were seen among the Clostridium difficile isolated from different animals. Clostridium sporogenes and Clostridium tertium also were isolated from some of the animals.