Article thumbnail
Location of Repository

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

By Bernard Lakaye, Alain Dubus, Bernard Joris and Jean-Marie Frère

Abstract

The outer membrane of gram-negative bacteria plays a major role in β-lactam resistance as it slows down antibiotic entry into the periplasm and therefore acts in synergy with β-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 β-lactamases. The method presented here uses the acylation of a highly sensitive periplasmic penicillin-binding protein (PBP) (BlaR-CTD) to assess the rate of β-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 β-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 × 10(−9) cm/s). Using these values, the MICs for a β-lactamase-overproducing strain of E. cloacae were successfully predicted, demonstrating the validity of the method

Topics: Analytical Procedures
Publisher: American Society for Microbiology
Year: 2002
DOI identifier: 10.1128/AAC.46.9.2901-2907.2002
OAI identifier: oai:pubmedcentral.nih.gov:127435
Provided by: PubMed Central
Download PDF:
Sorry, we are unable to provide the full text but you may find it at the following location(s):
  • http://dx.doi.org/10.1128/AAC.... (external link)
  • Suggested articles


    To submit an update or takedown request for this paper, please submit an Update/Correction/Removal Request.