A STUDY ON THE DISSOCIATION OF THE DIPHTHERIA BACILLUS

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Abstract
47 cultures of diphtheria bacilli isolated from cases showed S virulent forms only. Of 79 throat cultures isolated during convalescence, 16 showed presence of both S and R forms. 31 cases of clinical diphtheria were cultured at different stages from onset to release. Not a single R form was found during the acute stage, but 5 cases showed R types of diphtheria bacilli from cultures for release. In 3 cases of carriers studied, R forms were the predominating type. 1 culture of Park 8 diphtheria bacilli was dissociated into S and intermediate types by repeated plating on blood agar plates, and, while both forms produced toxin, the S type produced a much more potent toxin than the intermediate type. Sugar fermentations give reliable data for recognition of diphtheria bacilli, but do not differentiate S from R types, or virulent from avirulent bacilli. The virulence of 216 strains was tested. 42 were R forms and were all totally avirulent and atoxic. 164 were S forms, but, while 146 were virulent, 18 of them were avirulent. All the avirulent forms were isolated from convalescents or carriers. These 18 cultures demonstrate that while loss of toxicity is usually associated with the R cultural characteristics, a diphtheria culture may retain its S form and still have lost its toxic properties. Toxicity tests were made on 12 strains of R types and on 6 strains of R types transformed from S types. They were totally atoxic. Avirulent S forms were also found to be atoxic, while 3 strains of virulent S forms isolated from carriers were toxic, although the toxin was weak. 18 guinea pigs immunized with filtrates of R cultures showed no protection against filtrates of toxic S types. 11 guinea pigs immunized with R-type cultures showed no protection against S types of virulent bacilli. By repeated exposure of virulent cultures to 45[degree] C. for 17 hrs., virulence can be reduced, but this treatment has no effect on the appearance of the colonies. S virulent diphtheria bacilli inoculated into diphtheria antitoxin-broth mixtures and then into pure antitoxin showed no change after 20 passages, while 3 of 5 strains tested showed a change of S to R type after subculturing in anti-bacterial serum. Various attempts were made to transform R into S type again by growing the R type in toxin-broth mixture, by animal inoculation, and by subculturing in antibacterial serum of R diphtheria bacilli. By none of these methods was it possible to convert R back to S type. S virulent and toxic diphtheria bacilli are evidently transformed to non-virulent, R forms in the throats of patients during convalescence. The transformation may pass through an intermediate stage in which diminished toxin formation is not associated with morphological change. In vitro exps. indicate that the dissociation S [forward arrow] R is governed by contact with anti-bacterial rather than with antitoxic serum. Vaccination of carriers of S diphtheria bacilli with S vaccine is thus rendered logical.

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