The development of bacterial resistance to chloramphenicol was observed during a study of patients who were being treated with this drug for infections of the urinary tract (1). Nine of 33 strains of gram-negative bacilli isolated from the urine of 24 patients prior to therapy demonstrated during treatment a decrease in susceptibility to chloramphenicol. The development of resistance to the drug by some of these organisms was associated with therapeutic failures in five of these subjects. Similar observations had not been reported. Because of the potential clinical importance of these findings the results of a bacteriologic study of the strains which developed fastness to chloramphenicol in vivo, and quantitative observations on the origin of this phenomenon made in vitro are presented. METHODSThe strains of organisms that were studied were recovered from the urine of patients before, during, and after treatment with chloramphenicol. A description of cases and the schedules of treatment used in the clinical study have been noted elsewhere (1). Specimens of urine were obtained from females by catheterization and from males by careful local preparation prior to voiding. Ten cc. samples of urine were centrifuged and the sediment was stained for bacteria and cultured on MacConkey agar and on tryptose phosphate agar containing 5 per cent human blood. The various organisms that appeared were isolated in pure culture and were stored on tryptose phosphate agar slants at -200 C. In several instances agar pour plates of 10-fold dilutions of urine were made to determine the numbers of bacteria present. All strains of bacteria recovered from a single patient during all periods of observation were compared at the same time as regards their morphology, cultural and biochemical

Summary Six characteristics of the streptomycin-fast variants found in an antibiotic-sensitive strain of K. pneumoniae showed striking similarity to definitive properties of gene-mutations in higher organisms. Though circumstantial in nature, such evidence strongly favors the theory proposing the existance of mutable hereditary units in bacteria which quantitatively affect the property of streptomycin-susceptibility. Quantitatively, this stock strain was composed of a small number of organisms exhibiting low degrees of fastness to this antibiotic, and rare highly resistant cells in equilibrium with large numbers of sensitive organisms. The dynamics of this pattern of drug resistance appeared to be directed by factors that were inherent in each bacterial cell and its environment. When the latter was constant, the variant pattern was reproducible. A strain with a high degree of fastness to streptomycin was developed in a stepwise fashion when variants with a low degree of resistance were exposed to increasing concentrations of this antibiotic. This observation was best explained by the hypothesis of successive mutation and selection. Regardless of large concentrations of streptomycin, a highly fast strain appeared in a single step if the test population included rare but very resistant variants. The survival and establishment of streptomycin-fast variants at any given level of resistance appeared to be conditioned by factors concerned with the drug, the original environment, and the organism.