Permeability to cefsulodin of the outer membrane of Pseudomonas aeruginosa and discrimination between beta-lactamase-mediated trapping and hydrolysis as mechanisms of resistance

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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 thrid-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 .mu.M (1.25 .mu.g ml-1) and 37.6 .mu.M (20.0 .mu.g ml-1) respectively. The premeability 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 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 stiochiometry, would require the enzyme to be synthesized at 5.0 .mu.g 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.

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