Use of the fluorescent probe 1-N-phenylnaphthylamine to study the interactions of aminoglycoside antibiotics with the outer membrane of Pseudomonas aeruginosa

B. Loh, C. Grant, R. E.W. Hancock

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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 4-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 min 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 7 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 3 sites and demonstrated positive cooperativity. There was a statistically significant relationship between the pseudoassociation constant K(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.

Original languageEnglish
Pages (from-to)546-551
Number of pages6
JournalAntimicrobial Agents and Chemotherapy
Volume26
Issue number4
DOIs
Publication statusPublished or Issued - 1984
Externally publishedYes

ASJC Scopus subject areas

  • Pharmacology
  • Pharmacology (medical)
  • Infectious Diseases

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