title: Permeability to cefsulodin of the outer membrane of Pseudomonas aeruginosa and discrimination between beta-lactamase-mediated trapping and hydrolysis as mechanisms of resistance.
abstract: A pair of strains of Pseudomonas aeruginosa (3-Pre: cefsulodin-sensitive, inducible beta-lactamase; and 3-Post: cefsulodin-resistant, elevated beta-lactamase, derived from 3-Pre by subculture in the presence of cefsulodin) were taken as representative of the class of bacteria resistant to third-generation cephalosporins due to elevated synthesis of the normally inducible, chromosomally encoded beta-lactamase. These two strains were used to differentiate between 'trapping' and 'hydrolytic' mechanisms of cefsulodin resistance by (a) measuring the outer-membrane permeabilities to cefsulodin, (b) measuring the kinetics of cefsulodin hydrolysis and the stoichiometry of cefsulodin trapping by the periplasmic beta-lactamase, and (c) comparing the predictions of the trapping and hydrolysis hypotheses with the minimum inhibitory concentrations (MIC) of cefsulodin. The MIC of cefsulodin for strains 3-Pre and 3-Post were 2.35 microM (1.25 micrograms ml-1) and 37.6 microM (20.0 micrograms ml-1) respectively. The permeability parameter for cefsulodin of the outer membrane of the resistant strain was 0.0034 cm3 min-1 mg dry mass-1, so the flux of cefsulodin across its outer membrane at the MIC was calculated to be 0.120 nmol min-1 mg dry mass-1. Hydrolysis of cefsulodin by the beta-lactamase in the periplasm occurred at a rate of 0.118 nmol min-1 mg dry mass-1 which can thus account for resistance by matching the above rate of inflow. Trapping by the beta-lactamase, even with a 1:1 stoichiometry, would require the enzyme to be synthesized at 5.0 micrograms protein min-1 mg dry mass-1 or about 40% of the dry mass/generation. We conclude that hydrolysis, but not trapping, adequately explains the resistance to cefsulodin in P. aeruginosa 3-Post. A similar calculation for latamoxef resistance, using data taken from the literature, led to the same conclusion.
authors.affiliation: Department of Pathology and Bacteriology, University of Oxford, John Radcliffe Hospital, England.
authors.forename: R G
authors.initials: RG
authors.lastname: Hewinson
authors.forename: S J
authors.initials: SJ
authors.lastname: Cartwright
authors.forename: M P
authors.initials: MP
authors.lastname: Slack
authors.forename: R D
authors.initials: RD
authors.lastname: Whipp
authors.forename: M J
authors.initials: MJ
authors.lastname: Woodward
authors.forename: W W
authors.initials: WW
authors.lastname: Nichols
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism; D002441:Cefsulodin / Q000493:pharmacokinetics* / Q000494:pharmacology; D002463:Cell Membrane Permeability; D004351:Drug Resistance; D004790:Enzyme Induction / Q000187:drug effects; D006868:Hydrolysis; D007525:Isoelectric Focusing; D007700:Kinetics; D010957:Plasmids; D011550:Pseudomonas aeruginosa / Q000201:enzymology / Q000378:metabolism*; D001618:beta-Lactamases / Q000235:genetics / Q000378:metabolism*
chemical_list: D001425:Bacterial Outer Membrane Proteins; D001618:beta-Lactamases; D002441:Cefsulodin