Downloaded from www.microbiologyresearch.org by IP: 54.157.13.203 On: Mon, 08 Feb 2016 12:35:27 Journal of General Microbiology (1986), 132, 27-33. Printed in Great Britain 27 The Permeability Parameter of the Outer Membrane of Pseudumunas ueruginosa Varies with the Concentration of a Test Substrate, Cephalosporin C By R. GLYN HEWINSON,’ DAVID C. LANE,3 MARY P. E. SLACK’ AND WRIGHT W. NICHOLS2* Nufield Department of Pathology and Bacteriology, University of Oxford, Oxford, UK =Regional Public Health Laboratory, Level 6/7, John Radclifle Hospital, Oxford OX3 9DU. UK Centre for Mathematical Biology, Mathematical Institute, 24-29 St Giles, Oxford OX1 3LB, UK (Received 3 June 1985; revised 19 July 1985) The permeability parameter (C) for the movement of cephalosporin C across the outer membrane of Pseudomonas aeruginosa was measured using the widely accepted method of Zimmermann & Rosselet. In one experiment, the value of C varied continuously from 4-2 to 10.8 cm3 min-l (mg dry wt cells)-’ over a range of concentrations of the test substrate, cephalosporin C, from 50 to 5 PM. Dependence of C on the concentration of test substrate was still observed when the effect of a possible electric potential difference across the outer membrane was corrected for. In quantitative studies of Q-lactam permeation the dependence of C on the concentration of Q-lactam should be taken into account. INTRODUCTION The permeability of the outer membrane of Gram-negative bacteria is quantified as the ‘permeability parameter’, C (Zimmermann & Rosselet, 1977 ; Nikaido, 1979). The permeability parameter for a given p-lactam antibiotic is measured by determining the kinetics of hydrolysis of that antibiotic by intact cells and by broken cells of a P-lactamase-producing strain of the bacterium of interest; it is assumed to be constant under a particular set of experimental conditions and using a particular /3-lactam test substrate (Zimmermann & Rosselet, 1977; Nikaido, 1979). In this paper we demonstrate that this is not correct: the permeability parameter of intact cells of Pseudomonas aeruginosa varied with the concentration of a test substrate, cephalosporin C. PRINCIPLES OF THE MEASUREMENTS The assumption currently used for quantifying the permeability of the Gram-negative bacterial outer membrane is that the movement of a substance across this membrane obeys Fick’s laws of diffusion (Zimmermann & Rosselet, 1977). The steady state form of Fick’s first law in this context is J = DA(so - sp)/xp (Nikaido, 1979) where J is the flux of substrate across the outer membrane, D is the diffusivity of the substrate and A is the area available for diffusion; so and sp are the substrate concentrations in the exogenous water and the periplasmic space respectively, and xp is the distance down the diffusion pathway so that (sp - so)/xp is the concentration gradient. DA/xp is replaced by a single coefficient, C, the permeability parameter (Zimmermann & Rosselet, 1977) such that J = C(S, - sP) (1) The method of measuring C uses the presence of a cellular P-lactamase to hydrolyse P-lactam antibiotic in the periplasmic space. The rate of change of concentration of the fl-lactam 0001-2734 0 1986 SGM