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Coagulase-negative staphylococcus no. 7, isolated from the urogenital flora, was grown on semisolid brain heart infusion medium. Supernatants were obtained by centrifuging frozen and thawed media which had supported the growth of the staphylococci at 37 degrees C. The kinetic of production revealed that the antigonococcal activity was detected at the end of the logarithmic phase of growth and that the maximum activity was obtained after 24 h of incubation. Production of inhibitory activity was detected in cultures grown between 35 and 39 degrees C and in a pH range of 6.9 to 9.4. The inhibitory substance was purified by methanol extraction, acetone fractionation, dialysis, and chromatography on Ultrogel AcA 54. The characterization of the inhibitor showed that it was a lipoprotein or a lipid-associated-protein and that the protein component could be separated from the lipids when chromatographed on Ultrogel AcA 54 in the presence of urea. The inhibitory activity was associated with the protein component which had a molecular weight of approximately 15,900. In polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and urea, the protein component was dissociated in a subunit estimated to be approximately 1,400 daltons.

Twenty-four urogenital isolates of coagulase-negative staphylococci were selected because of their demonstrated ability to inhibit Neisseria gonorrhoeae growth in vitro. These organisms showed quantitative differences in their growth-interfering capability as revealed by a flip-flop agar overlay method. The composition of the culture medium affected the production of antigonococcal activity. Antigonococcal activity was shown with the following media: GC agar base with Lankford defined supplement, brain heart infusion agar, trypticase soy agar, and dextrose starch agar, but not with the GC agar base with CVA enrichment. An antigonococcal activity was obtained in the liquid phase prepared from semisolid agar cultures for 10 of the 24 staphylococcal isolates, whereas no activity was found in the supernatant from liquid cultures. The production of antigonococcal activity by staphylococci in vitro is influenced by growth conditions.

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