Shoki Nishida

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 104.1/g in young adults and 104.6/g in elderly adults, with a range of 102.0 to 106.9/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.

Sporulation and C2 toxin production by Clostridium botulinum type C strains producing no C1 toxin.

All of the 8 strains that were previously assumed to be nontoxigenic Clostridium botulinum type C were re‐examined for their toxigenicity and were demonstrated by trypsinization of the culture filtrates to produce C2 toxin under improved cultural conditions. One per cent glucose added to trypticase peptone medium enhanced C2 toxin production. The larger the spore population, the higher the C2 toxicity and when spore population was smaller than a level of 104/ml, no C2 toxicity was demonstrated. The C2 toxin was produced only during sporulation and not during vegetative growth.

Numerical Taxonomy of Saccharolytic Clostridia, Particularly Clostridium perfringens-Like Strains: Descriptions of Clostridium absonum sp. n. and Clostridium paraperfringens

Clostridium perfringens-like strains whose taxonomic position is uncertain were examined in detail. The lecithinases of these strains exhibited less avidity to the alpha-antitoxin of C. perfringens than did the C. perfringens lecithinase. On the basis of a computer analysis, the C. perfringens-like strains were grouped into two phenons, I and III, both of which are distinctly separable from C. perfringens (phenon II). Strains previously identified by us as belonging to Clostridium paraperfringens Nakamura et al. 1970, including strain G (= ATCC 27639), here designated as the type strain of C. paraperfringens, were found to belong to phenon I, and the strains of phenon III are regarded as constituting a new species, for which we propose the name Clostridium absonum. The type strain of C. absonum is HA-7103 (= ATCC 27555). The main characters differentiating C. absonum from C. perfringens are as follows: C. absonum produces a lecithinase which exhibits extremely low avidity to C. perfringens alpha-antitoxin, rapidly ferments salicin, does not ferment raffinose, and does not liquefy 10% gelatin; the main characters of C. absonum which differentiate it from phenon I strains are: Larger cell width, a distinctly stronger lecithinase reaction, liquefaction of 2% gelatin, production of butanol, and weak toxicity for mice. Deoxyribonucleic acid (DNA)-DNA homology studies of phenons I, II, and III confirmed the validity of the above-mentioned groupings. A computer analysis of other saccharolytic clostridia revealed that C. butyricum, C. acetobutyricum, and C. multifermentans are associated by similarity values higher than 90% and that C. septicum and C. chauvoei constitute separate, but closely related, taxa.

Cytotoxin production by Clostridium sordellii strains.

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% Na2HPO4 (m‐BHI) and cooked‐meat‐glucose (CMG) medium using baby hamster kidney (BHK‐21/WI‐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 difficile antitoxin neutralized C. sordellii cytotoxin and also C. sordellii antitoxin neutralized C. difficile cytotoxin.

Antimicrobial Susceptibility of Clostridium difficile from Different Sources

A total of 79 Clostridium difficile strains from healthy young and elderly adults, elderly patients without gastrointestinal disease, elderly patients receiving antibiotics without gastrointestinal complications, and elderly patients with antibiotic‐associated diarrhea or pseudomembranous colitis were tested for their susceptibilities to 24 antimicrobial agents. All of the 79 strains were inhibited by low concentrations of rifampicin, metronidazole, fusidic acid, vancomycin, ampicillin, and penicillin G. The strains were highly resistant to aminoglycosides, trimethoprim, sulfamethoxazole, nalidixic acid, and cycloserine and often resistant to neomycin, cefoxitin, and cefalexin. Wide variations in the susceptibility of C. difficile strains to erythromycin, clindamycin, lincomycin, chloramphenicol, and tetracycline were found. Strains resistant to erythromycin, clindamycin, and lincomycin were more frequently found among strains isolated from elderly adults than those isolated from young adults, with particularly high frequency among strains isolated from elderly patients receiving antibiotics. None of the 23 strains isolated from healthy young adults was resistant to chloramphenicol. All of the 14 strains resistant to erythromycin, clindamycin, lincomycin, and chloramphenicol were sensitive to tetracycline and all of the 15 strains resistant to erythromycin, clindamycin, lincomycin, and tetracycline were sensitive to chloramphenicol. Only one out of 19 tetracycline‐resistant strains was highly toxigenic, whereas 42 (70%) of 60 sensitive strains were highly toxigenic.

Germinability and Heat Resistance of Spores of Clostridium difficile Strains

Out of 111 Clostridium difficile strains, 108 produced spores in numbers of more than 105/ml and the remaining three did not produce any spores in brain heart infusion medium. The germination frequency in the medium without lysozyme varied widely from strain to strain, ranging from less than 10−8 to 100, and in 77 of the 108 strains the germination frequency was 10−5 or less. The spores, when treated with sodium thioglycollate and then inoculated into the medium containing lysozyme, germinated in all of the 108 strains at a frequency of 10−0.5 or more. The spores of two strains germinated at a frequency of more than 10−0.5 in all methods. Spores of C. difficile strains were fairly highly heat‐resistant; D100C values ranged from 2.5 to 33.5 min.

Effect of Clindamycin on Cytotoxin Production by Clostridium difficile

A total of 80 strains of Clostridium difficile, 33 toxigenic and 11 nontoxigenic clindamycin (CLDM)‐sensitive (MIC less than 12.5 μg/ml), and 23 toxigenic and 13 nontoxigenic CLDM‐resistant (MIC 200 to 6,400 μg/ml) 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.

Agglutination, toxigenicity and sorbitol fermentation of Clostridium difficile.

A total of 79 Clostridium difficile strains from different sources (50 strains from the fecal specimens of healthy adults, 13 from patients receiving antibiotics without gastrointestinal complications, 13 from antibiotic‐associated pseudomembranous colitis (PMC) or diarrhea patients, and three strains from ATCC) were investigated for agglutinability, using formol‐treated cells as antigen, in relation to toxigenicity. C. difficile strains tested were divided into four serovars, I, II, III, and IV, by the cross‐agglutination test. The agglutinin absorption test revealed that strains of serovar I, agglutinable with high titers (5,120–10,240) to antiserum prepared against a highly toxigenic C. difficile strain, ATCC 17859, possessed the serovar‐specific antigen. All of the strains of serovar I were highly toxigenic and all 13 strains isolated from the fecal specimens of antibiotic‐associated PMC or diarrhea patients belonged to this serovar, whereas 19 (38%) out of 50 strains from healthy adults and four (30.8%) out of 13 strains from patients receiving antibiotics without gastrointestinal complications possessed this antigen. None of the strains of other clostridial species than C. difficile were agglutinated by the three reference antisera used. Further study on the sugar fermentation test disclosed that the sorbitol‐fermenting property of C. difficile is very closely related to the toxigenicity and agglutinability.

Taxonomic relationships among Clostridium novyi Types A and B, Clostridium haemolyticum and Clostridium botulinum type C.

The present study was undertaken to examine the genetic relationships among the closely related species, Clostridium novyi types A and B, C. haemolyticum and C. botulinum type C. These species were tested for DNA-DNA homology and thermostability of DNA duplexes and sorted into three genetically related groups: I, C. novyi type A; II, C. novyi type B, C. haemolyticum and one C. botulinum type C strain (Stockholm); III, the remaining C. botulinum type C strains. A few biochemical criteria corresponding to the genetic differences were recommended to differentiate each group. These studies imply that C. haemolyticum might be considered as C. novyi type D and that there are two genetically different groups in C. botulinum type C.

Clostridium sporogenes Isolates and their Relationship to C. botulinum based on Deoxyribonucleic Acid Reassociation

Sixty-two isolates of Clostridium sporogenes from canned foods were examined for cultural properties, heat resistance and DNA-DNA homology to Clostridium botulinum type A190. Sporulation was observed in most of 21 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 II 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 II strains was 81% or more homologous to C. botulinum A190 DNA, forming duplexes which had thermostabilities similar to homologous duplexes of strain A190 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 A190 showed deltaT m(e) values of 7-0 degrees C when compared with the T m(e) of homologous DNA duplexes of strain A190.