Caroline A. Chandler

IN VITRO STUDIES OF POLYMYXIN

Studies of the in vitro activity of polymyxin have been in progress a t this laboratory since October, 1947. The preparation used is the hydrochloride of the partially purified concentrate, supplied by the Lederle Laboratories Division of the American Cyanamid Co., in 20-mg. ampoules labeled “B 71 Hydrochloride.” Observations have been made on the stability of the agent to heat and changes in the hydrogen ion concentration, its range of action and potency, and its effect upon the growth of Escherichia coli. The likelihood of the development of dmg fastneçs to polymyxin has been investigated. A method has been devised for assaying the concentration in body fluids and a search for antagonists has been started. EnvZronmentall+‘actors. The medium used in most of the tests about to be described was Difco Heart Infusion Broth with 0.05 per cent dextrose added, but the constitution of the medium appears to be unimportant in titrations with polymyxin. The end-points are not raised by the presence of serum in the tests. In fact, with serum they have averaged somewhat lower in 17 instances when comparisons were made. However, heating polymyxin in the presence of serum does result in a raising of the end-points as much as 8 times, the average in 20 tests being 4.3 times. On the other hand, the agent, at pH 7.2 in broth or salt solution, appears to be quite stable to heat, there being little or no change in activity after i t has stood at room temperature or in the 37°C. incubator for 18 hours, after heating at 56’C. for as much as 4 hours or after treatment in a boiling water bath for 10 minutes. Nevertheless, it keeps best, over long periods of time, in an acid environment in the deep freeze, -20°C. The loss of activity upon heating in the presence of serum is not only puzzling, but also inconvenieni. The sera of human beings and animals often contain antibodies for gram-negative bacteria which confuse the readings, and which must be eliminated by heating the sera a t 56’C. for 30 minutes. In assaying for polymyxin in the blood, i t is, therefore, essential to prepare a standard in serum and to heat it at the same time as the unknown sera. After heating, the standard and the unknown are diluted out in broth, in seria1 two-fold dilutions of 3 cc. each, and the tuhes are inoculated with

In vitro Studies of Aureomycin, a New Antibiotic Agent.

cc. of a 1:10,000 dilution af a 20-hour old culture of E. coli. The least amount of polymyxin which can be detected in this way is 0.62 to 1.25 pg./cc. The titrations are read a t 18 to 22 hours.

Studies on bacterial resistance to streptomycin.

Summary A new antibiotic, aureomycin, is effective in vitro against both gram positive and gram negative bacteria. Although its activity is of a lower order than that of penicillin for gram positive cocci or polymyxin for gram negative bacilli, it is effective against both classes of bacteria in concentrations close to those required of streptomycin. Inhibition at such concentrations, however, is fleeting in the case of aureomycin, much larger amounts being required for permanent suppression of growth. The new agent is bacteriostatic rather than bactericidal in its effect. Its antibacterial action is greatly diminished in the presence of serum, in vitro, and deterioration at room temperature is marked. Differences in the size of the inoculum have a moderate effect upon the minimal inhibitory concentration.

The Activity of Streptomycin in the Presence of Serum and Whole Blood.

Summary 1. Resistance to streptomycin can be induced by repeated transfer of various organisms in streptomycin containing media. 2. In the early logarithmic stage of growth, highly resistant mutants can be isolated, by chance selection, in a single transfer. 3. In most instances, acquired resistance to streptomycin is maintained. 4. Organisms resistant to streptomycin may retain their original virulence as measured by the bactericidal test. 5. The acquisition of resistance to streptomycin with the maintenance of virulence may have certain therapeutic implications.

Aureomycin; experimental and clinical investigations.

Summary 1. The activity of treptornycin on a susceptible strain of Staphylococczrs aureus was not changed in the presence of serum or whole blood. 2. The addition of fresh whole blood with or without immune serum did not augment streptomycin activity. 3. A resistant strain of Staph?dococcus aurcus was inhibited during the first 24 hours by streptomycin in low concentrations. This effect was augmented when serum was added to the medium. 4. Direct growth curves indicate that this inhibition by streptomycin ofa resistant strain was attributable to interference with growth of the organism rather than inactivation of labile constituents. 5. Phagocytosis in the presence of high concentrdtions of treptonlycin appears to be impaired. o. The bacteriostatic activity of streptomycin mas manifest on both susceptible and resistant strains. With the latter, thisinhibition was transitory. The bacteriostatic mechanisim and bactericidal mechanism aredissociated phenomena.