The role of the outer membrane of Pseudomonas aeruginosa in the uptake of aminoglycoside antibiotics

When cells of Pseudomonas aeruginosa were incubated with dihydrostreptomycin (DHS) for 30 sec then immediately washed with water, the amount of DHS adsorbed to the surface of the cells appeared to have been concentrated ‘intra-cellularly’ 312-fold as compared to the external medium. Working with isolated outer membranes (OM), it was found that 350 nmoles of DHS were adsorbed per mg of OM. The apparent dissociation constant was calculated to be 50 μM. Magnesium at concentrations of 170-fold higher than DHS prevented the adsorption of DHS to OM; but, the polyamines, spermine and spermidine, in concentrations equimolar to DHS, strongly inhibited the adsorption of DHS to the outer membrane. Tritiated DHS adsorbed to the cell surface could be displaced by DHS itself; by gentamicin; by magnesium; or by spermine. Gentamicin more effectively displaced DHS than DHS itself and in the reverse situation, DHS poorly replaced adsorbed gentamicin. Millimolar concentrations of magnesium were required to displace adsorbed DHS; but, only micromolar concentrations of spermine were required to achieve this same effect. Both magnesium and spermine inhibited all three phases of DHS uptake by intact cells. We propose that inhibition of the initial adsorption of DHS to the outer membrane by magnesium or spermine retarded the diffusion of this drug through the outer membrane. This action in turn would have a rate limiting effect on subsequent phases of uptake through the cell membrane. The cations, magnesium and the polyamines, acted both to prevent the adsorption of the aminoglycosides to the outer membrane of Ps. aeruginosa and to raise the minimal inhibitory concentrations of the antibiotics.