A pair of strains of Pseudomonas aeruginosa (3‐Pre: cefsulodin‐sensitive, inducible β‐lactamase; and 3‐Post: cefsulodin‐resistant, elevated β‐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 β‐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 β‐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 μM (1.25 μg ml−1) and 37.6 μM (20.0 μg 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 β‐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 β‐lactamase, even with a 1:1 stoichiometry, would require the enzyme to be synthesized at 5.0 μg 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.
|Nifer y tudalennau||9|
|Cyfnodolyn||European Journal of Biochemistry|
|Dynodwyr Gwrthrych Digidol (DOIs)|
|Statws||Cyhoeddwyd - 15 Chwef 1989|