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.

S U M M A R YSixty-two isolates of Clostridium sporogenes from canned foods were examined for cultural properties, heat resistance and DNA-DNA homology to Clostridium botulinum type ~1 9 0 .Sporulation was observed in most of 2 1 umbonate and rhizoidal colony-forming strains (colony-type I strains), but not in most of the 41 strains with convex and circular or crenate colonies with a mat to semi-glossy surface (colony-type I1 strains). More than half of the latter strains showed much higher heat resistance than the rhizoidal colony-forming strains. The DNA isolated from colony-type I1 strains was 81 % or more homologous to C. botulinum A I go DNA, forming duplexes which had thermostabilities similar to homologous duplexes of strain A I~O DNA. Colony-type I strains differed from C. botulinum by 30 to 40 % DNA homology and the DNA duplexes formed between these strains and strain ~1 9 0showed ATmce, values of 7.0 "C when compared with the Tfi,(,, of homologous DNA duplexes of strain ~1 9 0 .

~~Clostridium tetani and its related species C. tetanomorphum, C. cochlearium and C. lentoputrescens were examined for DNA-DNA homology and biochemical properties. Two distinctly different groups were included under the name of C. tetanomorphum: one was identical with C. cochlearium and the name C. tetanomorphum was applied to the other group with some amendment of biochemical properties. Comparison of the type strain of C. lentoputrescens with wild strains obtained from horse faeces indicated that the name C. lentoputrescens should be abolished as a later synonym of C. cochlearium. Liquefaction of gelatin and spore shape, which have been used as the important criteria for differentiation of C. tetani-related species, were genetically insignificant. I N T R O D U C T I O NThis study has investigated the relationships between Clostridium tetani-related species, i.e. C. tetani ( Bulloch et al. (1919) and the genus name Clostridium was applied to them in the first edition of Bergey's Manual of Determinative Bacteriology (Bergey et al., 1923). Since then, the name C. tetanomorphum has appeared in the Manual up to the seventh edition (Breed et al., 1957) but has disappeared from the eighth edition (Smith & Hobbs, 1974). Takahashi et al. (1967) have previously reported that two distinctly different groups were included under the name C. tetanomorphum. In the present study, tests for DNA-DNA homology, thermostability of DNA duplexes and biochemical properties were used to determine to which group the name of C. tetanomorphum should be applied. Furthermore, a possible identity of C. cochlearium and C. lentoputrescens has been re-investigated in the light of DNA-DNA homology determinations. METHODSStrains. These are listed, with the names as received, in Table 1. Strain K-130 was recovered from C. tetani HA-47 after heating at 100 "C for 30 min (Nishida et al., 1969). Non-toxigenic strains M-2, M-6 and M-14 have all the characteristics of C. tetani except toxicity.Preparation of DNA. Clostridium tetani HA-47, CN 655, 1110, 11 13, 1303, 61 14, 61 16, PO5 and 007, and C. tetani-like strain K-130 were grown at 37 "C in THPYG medium (pH 7.2) containing (%, w/v): trypticase (BBL), 0.5; heart infusion broth (Nissui, Tokyo, Japan), 0.5; polipeptone (Daigo, Osaka, Japan),