Novel method for measurement of outer membrane permeability to new β-lactams in intact Enterobacter cloacae cells

F. Bellido, J. C. Pechere, Robert Hancock

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The ability of five new β-lactams to permeate the outer membrane of intact Enterobacter cloacae β-lactamase-overproducing cells was measured by using a high-pressure liquid chromatography (HPLC)-based technique that avoided certain possible artifacts of the traditional methods. Low concentrations of antibiotics were mixed with bacterial suspensions, and at different times, the cells were removed from the medium by filtration. Residual β-lactam concentrations in the medium were then assessed by HPLC and UV detection. The assay was performed under conditions in which no β-lactamase activity was detected in the filtrate and the number of viable cells remained constant during the experiment. Outer membrane permeability was assessed with the Zimmermann-Rosselet equation, in which outer membrane permeability was rate limiting for hydrolysis of the β-lactam by periplasmic β-lactamase. Thus, the rate of disappearance of β-lactam was equal to the rate of outer membrane permeation. Preincubation of bacterial suspensions with 300 μg of cloxacillin per ml inhibited the hydrolysis of β-lactams by intact cells, demonstrating that β-lactam hydrolysis by periplasmic β-lactamase was essential in order to allow measurement of outer membrane permeability by this method. Permeabilitv coefficients (P) were calculated from the Zimmermann-Rosselet equation and were independent of the external concentration of antibiotic over a 100-fold concentration range. Cefepime and cefpirome exhibited rates of outer membrane permeation 5- to 20-fold higher than those of carumonam, ceftriaxone, and cefotaxime. Thus, the presence of a positive charge in the 3-lateral chain increased the permeation ability of β-lactam molecules considerably.

Original languageEnglish
Pages (from-to)68-72
Number of pages5
JournalAntimicrobial Agents and Chemotherapy
Issue number1
Publication statusPublished - 1 Jan 1991
Externally publishedYes

ASJC Scopus subject areas

  • Pharmacology
  • Pharmacology (medical)
  • Infectious Diseases

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