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Use of the fluorescent probe 1-N-phenyl napthylamine to study the interactions of aminoglycoside antibiotics with the outer membrane of Pseudomonas aeruginosa Loh, Bernadette Anne


The mode of interaction of the polycationic aminoglycoside antibiotics with the surface of Pseudomonas aeruginosa cells was studied using the hydrophobic fluorescent probe 1-N-phenyl napthylamine (NPN). Addition of the aminoglycoside gentamicin to intact cells in the presence of NPN led to a shift in the fluorescence emission maximum from 460 to 420 nm. At the same time the NPN fluorescence intensity increased four fold. Gentamicin caused no such effects when added to outer membrane vesicles suggesting that the increased fluorescence resulted from the interaction of gentamicin with intact cells. Gentamicin-promoted NPN uptake was inhibited by the divalent cations Mg²⁺ and Ca²⁺ , but occurred in the absence of gentamicin transport across the inner membrane. Low concentrations of gentamicin (2 μg/ml) caused NPN fluorescence to increase over a period of 4 minutes in a sigmoidal fashion. At higher concentrations (50 μg/ml) the increase occurred within a few seconds. The final fluorescence intensity was almost independent of the gentamicin concentration. A centrifugation technique was used to demonstrate that gentamicin caused actual uptake of NPN from the supernatant. The initial rate of NPN uptake varied according to the gentamicin concentration in a sigmoidal fashion. Similar data were obtained for seven other aminoglycoside antibiotics. The data when reanalysed as a Hill-plot gave a series of lines with a mean slope (the Hill number) of 2.26 ± 0.26, suggesting that the interaction of aminoglycosides with the cell surface to permeabilize it to NPN involved at least 3 sites and demonstrated positive cooperativity. There was a statistically significant relationship between the pseudo-association constant K[sub s] from the Hill plots and the minimal inhibitory concentrations for the 8 antibiotics These results are consistent with the concept that aminoglycosides interact at a divalent cation binding site on the P. aeruginosa outer membrane and permeabilize it to the hydrophobic probe NPN.

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