OXA-18, a class D clavulanic acid-inhibited extended-spectrum β-lactamase from Pseudomonas aeruginosa

Abstract

Clinical isolate Pseudomonas aeruginosa Mus showed resistance both to extended-spectrum cephalosporins and to aztreonam. We detected a typical double-disk synergy image when ceftazidime or aztreonam was placed next to a clavulanic acid disk on an agar plate. This resistance phenotype suggested the presence of an extended-spectrum β-lactamase. Isoelectric focusing revealed that this strain produced three β-lactamases, of pI 5.5, 7.4, and 8.2. A 2.6-kb Sau3A fragment encoding the extended-spectrum β-lactamase of pI 5.5 was cloned from P. aeruginosa Mus genomic DNA. This enzyme, named OXA-18, had a relative molecular mass of 30.6 kDa. OXA-18 has a broad substrate profile, hydrolyzing amoxicillin, ticarcillin, cephalothin, ceftazidime, cefotaxime, and aztreonam, but not imipenem or cephamycins. Its activity was totally inhibited by clavulanic acid at 2 μg/ml. Hydrolysis constants of OXA-18 (V(max), K(m)) confirmed the MIC results. Cloxacillin and oxacillin hydrolysis was noticeable with the partially purified OXA-18. The bla(OXA-18) gene encodes a 275-amino-acid protein which has weak identity with all class D β-lactamases except OXA-9 and OXA-12 (45 and 42% amino acid identity, respectively). OXA-18 is likely to be chromosomally encoded since no plasmid was found in the strain and because attempts to transfer the resistance marker failed. OXA-18 is peculiar since it is a class D β-lactamase which confers high resistance to extended-spectrum cephalosporins and seems to have unique hydrolytic properties among non-class A enzymes.