OXA-46, a New Class D β-Lactamase of Narrow Substrate Specificity Encoded by ablaVIM-1-Containing Integron from aPseudomonas aeruginosaClinical Isolate

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Abstract
A novel OXA-type enzyme, named OXA-46, was found to be encoded by a gene cassette inserted into a class 1 integron from a multidrug-resistantPseudomonas aeruginosaclinical isolate. The variable region of the integron also contained ablaVIM-1metallo-β-lactamase cassette and a duplicatedaacA4aminoglycoside acetyltransferase cassette. OXA-46 belongs to the OXA-2 lineage of class D β-lactamases. It exhibits 78% sequence identity with OXA-2 and the highest similarity (around 92% identity) with another OXA-type enzyme detected in clinical isolates ofBurkholderia cepaciaand in unidentified bacteria from a wastewater plant. Expression ofblaOXA-46inEscherichia colidecreased susceptibility to penicillins and narrow-spectrum cephalosporins but not to extended-spectrum cephalosporins, cefsulodin, aztreonam, or carbapenems. The enzyme was overproduced inE. coliand purified by two anion-exchange chromatography steps (approximate yield, 6 mg/liter). OXA-46 was made of a 28.5-kDa polypeptide and exhibited an alkaline pI (7.8). In its native form OXA-46 appeared to be dimeric, and the oligomerization state was not affected by EDTA. Kinetic analysis of OXA-46 revealed a specificity for narrow-spectrum substrates, including oxacillin, other penicillins (but not temocillin), and narrow-spectrum cephalosporins. The enzyme apparently did not interact with temocillin, oxyimino-cephalosporins, or aztreonam. OXA-46 was inactivated by tazobactam and carbapenems and, although less efficiently, also by clavulanic acid. Enzyme activity was not affected either by EDTA or by divalent cations and exhibited low susceptibility to NaCl. These findings underscore the functional and structural diversity that can be encountered among class D β-lactamases.

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