1) Significant reduction in antibacterial activity of chloramphenicol (CP) and dihydroxymethylfuratrizine (DMFZ) was observed in broth cultures of Clostridium with a series of 15 strains of the organisms covering 10 species. Such inactivation, however, could not be demonstrated with benzylpenicillin, cephaloridine, tetracycline and erythromycin. 2) Once they had lost their antibacterial activity in the broth, no discernible recovery of activity of CP or DMFZ was observed following removal of cells or heating (killing of vegetative forms of the organisms). 3) A maximum inactivation of CP under these conditions was obtained at 10 hours, and that of DMFZ at 16 hours following initation of incubation. 4) When loss of antibacterial activity of these agents was measured at several pH values, optimum ability of Clostridia to inactivate CP was observed at pH 6.0 to 9.0 and DMFZ at pH 7.0 to 9.0, respectively. 5) Optimum activity of Clostridia to reduce antibacterial activity of CP and DMFZ was observed between temperature of 40°C and 50°C and there was an evidence of diminution of inactivation at temperature above 60°C. 6) The loss of antibacterial activity of these two agents by Clostridia was evident under both anaerobic and aerobic conditions. 7) Reduction in antibacterial activity of CP and DMFZ did not occur in broth cultures of Clostridia from which cells had been removed. Suspension of washed cells in phosphate buffer exhibited significant reduction of the ability to inactivate CP or DMFZ. Resuspension, however, of these cells in broth resulted in recovery of the inactivating ability. 8) The MIC values of CP and DMFZ to the growth of Clostridia was found to vary widely with the size of the inoculum in the given assay system. It is evident from these results that Clostridia reduce the antibacterial activity of CP and DMFZ in broth culture. This ability of the organisms was deducible from the results of the present study to be reasonably attributed to some enzymatic system within the cells in a vegetative form.
The extraction of polysaccharide antigen, group specific antigen of Streptococcus, was carried out by the modified method of Slade. The antigen was extracted with 5% TCA at 90°C from whole dried cells and then aceton was added to the extraction. The polysaccharide, insoluble in aceton, was obtained as the precipitate. The precipitate was suspended in phenol-water and the insoluble material was discarded. By the addition of ethylalcohol to the phenol-water layer polysaccharide was obtained as the precipitate. The material was soluble in water and composed with 77% rhamnose, 18% hexosamine and 1.1% protein. As the sensitizing polysaccharide antigen O-stearoyl polysaccharide was used and the stearoyl polysaccharide contains 6% of the ester. The group specific antigenicity of the purified C-polysaccharide was demonstrated not only by the absorption test and gel diffusion technique but also by the stearoyl polysaccharide sensitized red cell hemagglutination.