The Role of Spontaneous Variants in the Acquisition of Streptomycin Resistance by the Shigellae

Abstract

The rate of development of streptomycin resistance of 12 strains of the Shigellae was detd. by 48-hr. broth subcultures in increasing streptomycin concs. All strains were initially inhibited by 3 to 7 U. of streptomycin and after 2 to 7 subcultures the majority of the strains grew in 1000 U. of streptomycin per ml., the highest conc., tested. Five of these re- sistant strains were subcultured daily for 50 days in broth; 2 showed no drop in resistance; 3 showed a significant drop in resistance. In order to determine the role of spontaneous variants in the development of streptomycin resistance 800 0.1-ml. samples from 20-24 hr. broth cultures of each of 6 original susceptible cultures (0.1 ml. of 20-24 hr. broth culture inhibited by 3-7 U. of streptomycin) were seeded into 800 tubes containing 1000 U. of streptomycin per ml. From each of the 6 susceptible cultures bacteria were obtained capable of growing directly in 1000 U. of streptomycin. Using a procedure which permitted the continuous selection of susceptible streptomycin colonies it was found that the susceptible colonies were still capable of giving rise to a small number of highly resistant streptomycin variants. These variants could only be detected by examining large numbers of bacteria as indicated above. Washed resting bacteria though exposed to active streptomycin concns. for 7 days at varying temps. failed to show any adaptation to increased streptomycin cones. It is concluded that the in vitro development of streptomycin resistance results from the development of resistant variants independent of the direct action of streptomycin, followed by the selection of these variants in a high streptomycin conc. A single resistant variant is sufficient to cause a high degree of streptomycin resistance.