Abstract
The mode of interaction of the polycationic aminoglycoside antibiotics with the surface of Pseudomonas aeruginosa cells was studied with the hydrophobic fluorescent probe 1-N-phenylnaphthylamine (NPN). The 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 fourfold. 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 micrograms/ml) caused NPN fluorescence to increase over a period of 4 min in a sigmoidal fashion. At higher concentrations (50 micrograms/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 reanalyzed 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 three sites and demonstrated positive cooperativity. There was a statistically significant relationship between the pseudoassociation constant K, from the Hill plots and the minimal inhibitory concentrations for the eight antibiotics. These results are consistent with the concept that aminoglycosides interact as a divalent cation binding site on the P. aeruginosa outer membrane and permeabilize it to the hydrophobic prove NPN.
Highlights
The aminoglycosides are a group of polycationic antibiotics first recognized in the 1940s with the discovery of streptomycin
In this paper we have demonstrated the effectiveness of fluorescent probes in studying the interactions of aminoglycosides with P. aeruginosa cells
M.Sc. thesis, University of British Columbia, 1984). These vesicles became immediately saturated with NPN that had fluorescence spectral properties similar to the NPN taken up by gentamicin-treated cells. This suggests that gentamicin is disorganizing the cell surface in such a way that NPN can partition into the outer membrane
Summary
The aminoglycosides are a group of polycationic antibiotics first recognized in the 1940s with the discovery of streptomycin. The divalent cation cross-bridging of lipopolysaccharide is disrupted, causing localized destabilization or distortion of the membrane [6] In support of this hypothesis, the interaction of streptomycin or gentamicin with P. aeruginosa causes enhancement of outer membrane permeability to lysozyme and increased uptake of the ,B-lactam nitrocefin [7]. EDTA, which disrupts Mg2+ cross-bridges by chelation rather than displacement, causes similar enhancement of uptake of lysozyme and nitrocefin [7] as well as an increased rate of killing by aminoglycosides [17] Another line of evidence is that outer membrane-altered mutants of P. aeruginosa, with an apparent decrease in the number of Mg-binding sites, show cross-resistance to EDTA, polymyxin, and aminoglycosides [13].
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