‘Covalent trapping’ and latamoxef resistance in β-lactamase-derepressed Pseudomonas aeruginosa

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Abstract
Three Pseudomonas aeruginosa strains which constitutively produced chromosomal (Id, or Sabath and Abraham) β-lactamase in large amounts were resistant to latamoxef (moxalactam) MICs, 128–256 mg/l). Their β-lactamase-basal mutants, which produced 1200–18,000-fold less enzyme, were latamoxef-sensitive (MICs, 4–16 mg/l), suggesting that the enzyme caused the resistance of the parent organisms. Latamoxef was a feeble substrate of the enzyme (kcal <0.5/min) but reacted to form a stable complex that lacked catalytic activity against benzylpenicillin. The complex was isolated by gel filtration and was shown to be stable to isoelectric focusing, suggesting a covalent link between the enzyme and latamoxef. During incubation the complex underwent a slow breakdown, regenerating active enzyme. This breakdown obeyed first-order kinetics, and the half-life of the inactivated form was 19±1min at 37°C. Binding of antibiotic molecules in this complex may contribute to the latamoxef-resistance observed in the β-lactamase-derepressed strains. This ‘covalent trapping’ should be distinguished from the ‘non-covalent trapping’ proposed elsewhere as a general mechanism of β-lactamase-mediated resistance to reversibly-bound weak-substrate β-lactams.