An antagonist of streptomycin and dihydrostreptomycin produced by Pseudomonas aeruginosa.

SUMMARY: A substance produced by Pseudomonas aeruginosa (P. pyocyanea) which antagonizes the activity of streptomycin and dihydrostreptomycin, and which will itself inhibit bacterial growth in low concentrations, has been isolated in a crystalline form. The antagonist at 0.01 μg./ml. was capable of antagonizing the action of 1000 times its weight of dihydrostreptomycin against Bacillus subtilis or B. pumilus. It was much less active against Escherichia coli. Growth of B. subtilis and B. pumilus was inhibited by less than 1 μg. antagonist/ml. under conditions which required a concentration of 6 μg. dihydrostreptomycin sulphate/ml. to inhibit growth. A method of assay of antagonist activity, capable of assaying solutions containing 0.01 μg. antagonist/ml., was developed. It was a cup-plate method which depended on the production of zones of exhibition. Plates poured with seeded nutrient agar containing an inhibitory concentration of dihydrostreptomycin were used, and the samples of antagonist were placed on the surface of the agar in fish spine beads. The zones of exhibition developed during incubation of the plates and the potency of the samples could be determined from a comparison with zones produced by a standard preparation on the same plate. The antagonist was produced by most strains of Pseudomonas aeruginosa under favourable conditions. Its production in fluid culture depended on vigorous aeration, but too high an aeration rate or foaming of the culture produced low yields. Strains of other organisms which were examined, for example, Staphylococcus aureus, Escherichia coli, Serratia marcescens, Bacterium bodenheimer, streptococci of Lance-field's Group D, Bacillus subtilis and B. pumilus, did not produce antagonist. The antagonist was extracted from fluid culture by n-butanol; the butanol was removed by distillation in an atmosphere of nitrogen, and the residue fractionated by precipitation from benzene solution by addition of light petroleum. The success of the fractionation was dependent on obtaining an initial butanol extract containing at least 100 μg. antagonist/mg. total solids. This in turn was dependent on the use of a simple chemically defined culture medium for production, and the choice of a strain of Pseudomonas aeruginosa which produced a high yield of antagonist. The crystalline antagonist has the formula C17–18H23–24NO2 and a molecular weight of the order 300–400. It is stable in the dry crystalline state, but readily oxidized in solution. It is soluble in ethanol and butanol, less soluble in benzene, and relatively insoluble in light petroleum. Its maximum solubility in water at room temperature at pH 7.0 is c. 10 μg./ml.; at pH 9.0 it is 20–30 μg./ml.