SURFACE ACTION OF GENTAMICIN ON PSEUDOMONAS-AERUGINOSA

The mode of action of gentamicin has traditionally been considered to be at the 30S ribosomal level. However, the inhibition of bacterial pr otein synthesis alone appears to be insufficient to entirely explain t he bactericidal effects. Bacteriolysis is also mediated through pertur bation of the cell surface by gentamicin (J. L. Kadurugamuwa, J. S. La m, and T. J. Beveridge, Antimicrob. Agents Chemother. 37:715-721, 1993 ). In order to separate the surface effect from protein synthesis in P seudomonas aeruginosa PAO1, we chemically conjugated bovine serum albu min (BSA) to gentamicin, making the antibiotic too large to penetrate through the cell envelope to interact with the ribosomes of the cytopl asm. Furthermore, this BSA-gentamicin conjugate was also used to coat colloidal gold particles as a probe for electron microscopy to study t he surface effect during antibiotic exposure. High-performance liquid chromatography confirmed the conjugation of the protein to the antibio tic. The conjugated gentamicin and BSA retained bactericidal activity and inhibited protein synthesis on isolated ribosomes in vitro, but no t on intact cells in vivo because of its exclusion from the cytoplasm. When reacted against the bacteria, numerous gentamicin-BSA-gold parti cles were clearly seen on the cell surfaces of whole mounts and thin s ections of cells, while the cytoplasm was devoid of such particles. Di sruption of the cell envelope was also observed since gentamicin-BSA a nd gentamicin-BSA-gold destabilized the outer membrane, evolved outer membrane blebs and vesicles, and formed holes in the cell surface. The morphological evidence suggests that the initial binding of the antib iotic disrupts the packing order of lipopolysaccharide of the outer me mbrane, which ultimately forms holes in the cell envelope and can lead to cell lysis. It is apparent that gentamicin has two potentially let hal effects on gram-negative cells, that resulting from inhibition of protein synthesis and that resulting from surface perturbation; the tw o effects in concert make aminoglycoside drugs particularly effective antibiotics.