Reference : Method for estimation of low outer membrane permeability to beta-lactam antibiotics
Scientific journals : Article
Life sciences : Microbiology
Human health sciences : Pharmacy, pharmacology & toxicology
Method for estimation of low outer membrane permeability to beta-lactam antibiotics
Lakaye, Bernard mailto [Université de Liège - ULg > Département des sciences biomédicales et précliniques > Biochimie et physiologie humaine et pathologique >]
Dubus, Alice mailto [Université de Liège - ULg > Département de chimie (sciences) > Département de chimie (sciences) >]
Joris, Bernard mailto [Université de Liège - ULg > > Centre d'ingénierie des protéines >]
Frère, Jean-Marie mailto [Université de Liège - ULg > Département des sciences de la vie > Département des sciences de la vie >]
Antimicrobial Agents and Chemotherapy
Amer Soc Microbiology
Yes (verified by ORBi)
[en] The outer membrane of gram-negative bacteria plays a major role in beta-lactam resistance as it slows down antibiotic entry into the periplasm and therefore acts in synergy with beta-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 beta-lactamases. The method presented here uses the acylation of a highly sensitive periplasmic penicillin-binding protein (PBP) (BlaR-CTD) to assess the rate of beta-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 beta-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 beta-lactamase-overproducing strain of E. cloacae were successfully predicted, demonstrating the validity of the method.

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