Biochemical properties of Clostridium difficile were reinvestigated for the practical identification of the organism in clinical laboratories. Bacterial growth in 2% proteose peptone medium supplemented with 0.01 % r-cysteine Hel and 0.1 % agar supported sufficient growth to read the fermentation results just as well as did pre-reduced anaerobically sterilized medium. Incubation for 2 days was long enough for determining the ability to ferment fructose, glucose, mannitol, mannose, melezitose, and sorbitol. All of the 82 strains liquefied 2% but not 10% gelatin. The significance of mannitol fermentation and gelatin liquefaction is stressed since C. difficile is the only species fermenting mannitol among the gelatin-liquefying species of clostridia having subterminal spores.It is now recognized that Clostridium dijJicile is a major cause of antibiotic-associated pseudomembranous colitis of man (l). The carbohydrate-fermenting properties of C. dijJicile were described by Hall and O'Toole when they first isolated this microbe in 1935 (4). Recently, further detailed information about the carbohydrate-fermenting properties of this microbe has been reported by several investigators. Hafiz and Oakley (3) used proteose peptone broth as a basal medium for the carbohydrate fermentation test and incubated cultures anaerobically at 37 C for a maximun of 40 days, which is too long for practical purposes, while Holdeman et al (5) determined the properties by incubating the cultures in prereduced anaerobically sterilized (PRAS) medium at 37 C for 24 to 72 hr. The present paper describes the biochemical properties of C. dijJicile determined by a simple method for practical purposes, disclosing that PRAS medium is not necessary for the test. MATERIALS AND METHODSBacterial strains. A total of 82 C. dijJicile strains, 56 toxigenic and 26 nontoxigenic, were employed. Most of these strains were isolated from healthy adults and

A total of 80 strains of Clostridium difficile, 33 toxigenic and 11 nontoxigenic clindamycin (CLDM)-sensitive (MIC less than 12.5 p,gJml) , and 23 toxigenic and 13 nontoxigenic CLDM-resistant (MIC 200 to 6,400 p,gJml) were tested for cytotoxin production in the presence of CLDM. None of the 24 nontoxigenic strains produced cytotoxin regardless of the presence of CLDM and only six out of the 56 toxigenic strains showed 16-to 64-fold higher levels of cytotoxic activity in the presence of CLDM at the concentrations of 1/2 to 1/32 of the MIC than in the absence of CLDM; all of the six strains were CLDM sensitive. Further studies revealed that addition of CLDM to the culture caused enhanced cytotoxin synthesis, and that the maximum production of cytotoxin was obtained when CLDM was added to the medium at the time of inoculation or of the ensuing early logarithmic phase. Also, the influence of other antibiotics on the effect of CLDM was examined. Addition of metronidazole, vancomycin, chloramphenicol, cephaloridine, or penicillin, which induced cytotoxin to medium containing CLDM did not increase the effect of CLDM any further. Addition of CLDM to medium containing tetracycline, which inhibited cytotoxin production, induced cytotoxin production but not fully.Toxin-producing strains of Clostridium difficile in the fecal flora have been incriminated as a causative agent in antibiotic-associated pseudomembranous colitis in humans and cytotoxin in feces has been shown to be a good marker for this disease (1), although discrepancies between cytotoxin titers obtained by tissue culture assay and severity of the disease have been noted (13).Recently it was stated that a certain other toxin rather than cytotoxin, both of which are produced by the vast majority of C. difficile strains, might play an important role in antibiotic-induced colitis (12). The detection of cytotoxin in feces, however, seems to be worth using for diagnosing antibiotic-associated pseudo. membranous colitis, because of the good agreement between the presence of cytotoxin in the feces and the involvement of this disease (1). Cytotoxin production in the gastrointestinal tract seems to be affected by the presence of antibiotics,

A total of 55 strains of Clostridium sordellii, 21 lethal toxin-positive and 34 lethal toxin-negative, were tested for cytotoxin production in brain heart infusion medium supplemented with 0.2% Na2HP04 (m-BHI) and cooked-meat-glucose (CMG) medium using baby hamster kidney (BHK-21jWI-2) cells as indicator cells. The m-BHI medium was preferred to CMG medium and 24 hr of incubation was sufficient for cytotoxin production. Nineteen of the 21 toxigenic strains were also cytotoxigenic, and the strength of the cytotoxigenicity was approximately parallel with that of the lethal toxigenicity. Clostridium difJicile antitoxin neutralized C. sordellii cytotoxin and also C. sordellii antitoxin neutralized C. difJicile cytotoxin.

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In the previous paper (5), the authors indicated that some strains of Clostridium sordellii produced a toxin causing rounding of tissue culture cells. In the present study, C. sordellii strains were examined for ileal loop response on the basis of recent findings of close relationships between cytotoxicity and ileal loop response found in other clostridia (2,3,6,7).A total of 49 C. sordellii strains, 15 cytotoxigenic and 34 non-cytotoxigenic, were used (5). A O.l-ml portion of an overnight culture of each strain in liver broth was inoculated into 10 ml of brain heart infusion medium (BBL, Cockeyville) supplemented with 0.2% Na2HP04 (m-BHI medium). The inoculated medium was incubated at 37 C under anaerobic conditions (80% nitrogen, 10% carbondioxide, and 10% hydrogen). After incubation for 24 hr, the culture was centrifuged at 3,500 xg for 5 min. The supernatant was filtered through a membrane filter with a 0.45-pm pore size (Japan-Millipore, Tokyo), and used for the cytotoxicity assay and ileal loop test.Ligated intestinal loops (seven loops per rabbit with each loop 7 to 10 em in length) were prepared in Japanese white rabbits (1.7 to 2.0 kg) according to the method of De and Chatterje (I). Rabbits were fasted for 24 hr before surgery, and water was provided ad lib before and after surgery. One to 8 ml of each culture filtrate was injected into the ends of loops and the injection site was separated from the remaining portion of the loop by ligation to prevent leakage of the sample. The rabbits were sacrificed 20 hr after the ligation. Each extirpated loop was examined for the ratio of its fluid volume (ml) to length (em). Specimens obtained from loops with positive or negative response were fixed in 20% buffered formalin (pH 7.0) and embedded in paraffin. The 4 pm thick sections were stained with hematoxylin and eosin for microscopic observation.Cytotoxicity was assayed according to the previously described microtiter method (4) on monolayers of baby hamster kidney (BHK-21/WI-2) cells. The reciprocal of the highest dilution causing complete rounding of the cells was used as the number of cytotoxic units (CD) 150 pI of sample.Ileal loop responses of all strains tested are summarized in Table I. When I ml of original culture filtrate of strain 3703 or its two-fold dilution was injected, the rabbits died. Therefore, I ml of the four-fold diluted culture filtrate of 807