Isolation and partial characterization of Bac201: A plasmid-associated bacteriocin-like inhibitory substance from Staphylococcus aureus AB201

Abstract

Staphylococcus aureus AB201, a clinical isolate from wound pus, produced a bacteriocin-like inhibitory substance termed as Bac201 that exhibited a broad-spectrum activity against both gram-positive as well as gram-negative bacteria. Among gram-negative bacteria it was active against Neisseria meningitidis and Acinetobacter calcoaceticus both being gram-negative cocci. Inhibition due to the effect of organic acids, hydrogen peroxide, or bacteriophages was excluded. This inhibitory substance could not be induced or eluted. It was partially purified to 80% saturation by ammonium sulfate precipitation resulting in maximum specific activity of 829 AU/mg (25-fold increase). Proteolytic enzymes rapidly inactivated the antagonistic activity of the partially purified material, whereas glycolytic and lipolytic enzymes had no effect. It remained stable in the presence of mild organic solvents. It could be stored at -20 °C without loss of activity, stable at 60 °C and 80 °C for 30 min, 100 °C for 20 min and autoclaving temperature (121 °C for 15 min), and exhibited activity within a wide range of pH (2.5-10). Bac201 had an estimated M(r) of 41kDa, as indicated by activity detection after SDS-PAGE. Temperature-mediated (44 °C) plasmid curing studies suggested linkage of bacteriocin production to a 4.8 MDa plasmid. The Bac201 was bactericidal rather than bacteriolytic.