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

The ability of five new beta-lactams to permeate the outer membrane of intact Enterobacter cloacae beta-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 beta-lactam concentrations in the medium were then assessed by HPLC and UV detection. The assay was performed under conditions in which no beta-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 beta-lactam by periplasmic beta-lactamase. Thus, the rate of disappearance of beta-lactam was equal to the rate of outer membrane permeation. Preincubation of bacterial suspensions with 300 micrograms of cloxacillin per ml inhibited the hydrolysis of beta-lactams by intact cells, demonstrating that beta-lactam hydrolysis by periplasmic beta-lactamase was essential in order to allow measurement of outer membrane permeability by this method. Permeability 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 beta-lactam molecules considerably.