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

Abstract

A pair of strains of Pseudomonas aeruginosa (3-Pre : cefsulodin-sensitive, inducible /?-lactamase ; and 3-Post: cefsulodin-resistant, elevated fl-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 j-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 b-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 pM (1.25 pg ml-') and 37.6 pM (20.0 pg ml-') respectively. The permeability parameter for cefsulodin of the outer membrane of the resistant strain was 0.0034 cm3 min-' mg dry mass-', so the flux of cefsulodin across its outer membrane at the MIC was calculated to be 0.120 nmol min-' mg dry mass-'.Hydrolysis of cefsulodin by the p-lactamase in the periplasm occurred at a rate of 0.1 18 nmol min-' mg dry mass-' which can thus account for resistance by matching the above rate of inflow. Trapping by the P-lactamase, even with a 1 : 1 stoichiometry, would require the enzyme to be synthesized at 5.0 pg protein min-' mg dry mass-' 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.Resistance to third-generation cephalosporins [l] in gramnegative bacteria possessing inducible class-C [2] P-lactamases can occur by mutation to continuous elevated synthesis of the enzyme [3 -91. The mechanism of this form of resistance has been the subject of much debate, because in general these p-lactamases possess low or undetectable hydrolytic activity against such compounds [8, proposed that the mechanism of resistance was one of trapping (i. Abbreviations. CDM, chemically defined medium; MIC, minimum inhibitory concentration.Enzymes. fi-Lactamase (EC 3.5.2.6); lysozyme (EC 3.2.1.17); ribonuclease A (EC 3.1.27.5).In order to test the hypothesis that a low hydrolytic activity is sufficient to explain the antibiotic resistance, three sets of measurements need to be made [19-221. The first is the MIC (minimum inhibitory concentration) of a third-generation cephalosporin for a ,U-lactamase-inducible strain, and for an otherwise isogenic resistant mutant synthesizing an elevated amount of fl-lactamase. Secondly the kinetics of hydrolysis and the stoichiometry of trapping of the compound by the j-lactamase in each of the above two strains must be measured. Thirdly, the outer membrane permeability of each of the above ...

Outer membrane permeability and beta-lactamase stability of dipolar ionic cephalosporins containing methoxyimino substituents

“…With E. cloacae, the permeability coefficients obtained tended to be higher with cells containing higher levels of 1-lactamase, suggesting that some enzyme molecules may be present on the cell surface (3). Because of this problem, permeability was determined with the uninduced wild-type strain 55, but even these values should be considered maximal estimates.…”

“…Thus, there is no need to * Corresponding author. assume a resistance mechanism based on nonhydrolytic binding or trapping (9,10), and this was found to be true even with one of the most slowly hydrolyzed compounds, moxalactam, and with a bacterium with one of the least permeable outer membranes, P. aeruginosa (3).…”

Separation and structural elucidation of cefsulodin and its impurities in both positive and negative ion mode in cefsulodin sodium bulk material using liquid chromatography/tandem mass spectrometry

“…Cefsulodin sodium can specifically bind to penicillinbinding protein 1B and inhibit the synthesis of peptidoglycan in the bacterial cell wall, resulting in bacterial death due to the expansion and lysis of cell wall defects. [3][4][5][6][7] In clinical drugs, impurity control is an important factor since impurities may reduce antibacterial activity, increase toxicity and affect clinical efficacy. Moreover, impurities are also formed in the storage and transportation process.…”