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Antimicrobial Agents and Chemotherapy, September 2002, p. 2901-2907, Vol. 46, No. 9
0066-4804/02/$04.00+0     DOI: 10.1128/AAC.46.9.2901-2907.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Method for Estimation of Low Outer Membrane Permeability to ß-Lactam Antibiotics

Bernard Lakaye,,{dagger} Alain Dubus, Bernard Joris, and Jean-Marie Frère*

Laboratoire d'Enzymologie and Centre d'Ingénierie des Protéines, Université de Liège, Institut de Chimie, B-4000 Liège, Belgium

Received 19 October 2001/ Returned for modification 7 February 2002/ Accepted 21 May 2002

The outer membrane of gram-negative bacteria plays a major role in ß-lactam resistance as it slows down antibiotic entry into the periplasm and therefore acts in synergy with ß-lactamases and efflux systems. Up to now, the quantitative estimation of low outer membrane permeability by the method of Zimmermann and Rosselet was difficult because of the secreted and cell surface-associated ß-lactamases. The method presented here uses the acylation of a highly sensitive periplasmic penicillin-binding protein (PBP) (BlaR-CTD) to assess the rate of ß-lactam penetration into the periplasm. The method is dedicated to measurement of low permeability and is only valid when the diffusion rate through the outer membrane is rate limiting. Cytoplasmic membrane associated PBPs do not interfere since they are acylated after the very sensitive BlaR-CTD. This method was used to measure the permeability of ß-lactamase-deficient strains of Enterobacter cloacae and Enterobacter aerogenes to benzylpenicillin, ampicillin, carbenicillin, cefotaxime, aztreonam, and cephacetrile. Except for that of cephacetrile, the permeability coefficients were equal to or below 10-7 cm/s. For cephacetrile, carbenicillin, and benzylpenicillin, the outer membrane of E. cloacae was 20 to 60 times less permeable than that of Escherichia coli, whereas for cefotaxime, aztreonam, and ampicillin it was, respectively, 400, 1,000, and 700 times less permeable. The permeability coefficient for aztreonam is the lowest ever measured (P = 3.2 x 10-9 cm/s). Using these values, the MICs for a ß-lactamase-overproducing strain of E. cloacae were successfully predicted, demonstrating the validity of the method.

* Corresponding author. Mailing address: Laboratoire d'Enzymologie and Centre d'Ingénierie des Protéines, Université de Liège, Institut de Chimie, B6 Sart-Tilman, B-4000 Liège, Belgium. Phone: 32/4/366.33.98. Fax: 32/4/366.33.64. E-mail: jmfrere{at}ulg.ac.be.

{dagger} Present address: Centre de Recherches en Neurobiologie Cellulaire et Moleculaire, Université de Liège, B-4020 Liège, Belgium.

Antimicrobial Agents and Chemotherapy, September 2002, p. 2901-2907, Vol. 46, No. 9
0066-4804/02/$04.00+0     DOI: 10.1128/AAC.46.9.2901-2907.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.





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