Development of bacterial resistance to streptovaricin.

Streptovaricin' is a complex of closely related antibiotics which has received extensive study in our laboratories because of its broad in vitro antibacterial spectrum, and its in vivo activity against experimental infections of mice and guinea pigs with virulent strains of Mycobacterium tuberculosis (Siminoff et al., 1957; Whitfield et al., 1957; Rhuland et al., 1957). A number of interesting phenomena relating to the antibacterial effect of the drug have been observed. In a standard tube-dilution test for bacterial inhibition, end point shifts occurred when the 16-hr incubation period was extended to 40 hr. Subculture of the bacteria from the overgrown tubes into a fresh dilution series indicated either sensitivity equal to that of the parent culture, or apparent chance development of moderate to extremely high resistance to streptovaricin. Another observation has been the occurrence of "skips" in a dilution series, i. e., occasional growth in tubes containing concentrations of streptovaricin in excess of the minimal inhibitory concentration. Such skips appeared frequently when a 20-hr culture of Staphylococcus aureus was inoculated at 10-3 dilution into drug-containing media, but were almost never seen at an inoculum dilution of 10-4 6. These observations raised a number of questions: (a) Is the development of resistance in the tube-dilution test due to spontaneous mutation or to an adaptation phenomenon? (b) What is the mutation rate? (c) What is the nature of the development of resistance? and (d) What is the basis for the effect of inoculum size upon the antibacterial activity of streptovaricin? To attempt to answer these questions, we have employed the Luria-Delbriick bacterial fluctuation test (Luria and Delbrtick, 1943) and have made quantitative studies of the growth of relatively small and large populations of S. aureus in the presence and absence of streptovaricin and have analyzed these populations for the emer1 The Upjohn Company trademark name is Dalacin. gence of resistant cells. However, these experiments were complicated by the instability of streptovaricin. When the antibiotic is incubated in the test medium, brain heart infusion broth (Difco) adjusted to pH 6.8 at 37 C, from 40 to 70 per cent of the activity is lost after 16 hr, and 90 per cent by 40 hr (Siminoff et al., 1957). Therefore, the resistant cells were analyzed for stability of the resistance character and extent of resistance development by the replica plating method (Lederberg and Lederberg, 1952).