Production, purification, and preliminary characterization of a gonococcal growth inhibitor produced by a coagulase-negative staphylococcus isolated from the urogenital flora

Abstract

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.