OXA-24, a novel class D beta-lactamase with carbapenemase activity in an Acinetobacter baumannii clinical strain

Abstract

Acinetobacter baumannii RYC 52763/97, a clinical isolate involved in a prolonged nosocomial outbreak at our hospital, was resistant to all beta-lactams tested, including imipenem and meropenem, which had MICs of 128 and 256 microg/ml, respectively. This strain synthesized three beta-lactamases: a plasmid-mediated TEM-1 beta-lactamase (pI 5.4), an AmpC-type chromosomal cephalosporinase (pI 9.4), and a novel, presumptively chromosomally mediated OXA-related enzyme (pI 9.0) named OXA-24. After cloning and sequencing, the deduced amino acid sequence of the OXA-24 beta-lactamase showed 40% homology with the OXA-10 (PSE-2) and OXA-7 beta-lactamases, 39% homology with the OXA-11 and OXA-5 enzymes, and 33% homology with the LCR-1 beta-lactamase. The amino acid sequence of the OXA-24 beta-lactamase contained the STFK motif found in serine beta-lactamases, but the typical class D triad KTG was replaced by KSG and the motif YGN was replaced by FGN. The OXA-24 beta-lactamase hydrolyzed benzylpenicillin and cephaloridine but lacked activity against oxacillin, cloxacillin, and methicillin. The enzymatic activity was inhibited by chloride ions and by tazobactam (50% inhibitory concentration [IC(50)], 0.5 microM), sulbactam (IC(50), 40 microM), and clavulanic acid (IC(50), 50 microM). Carbapenem MICs for an Escherichia coli transformant (pBMB-1) expressing the cloned OXA-24 enzyme had a fourfold increase. Relative V(max)/K(m) values of 13 and 6 were obtained with imipenem and meropenem, respectively, and a positive microbiological assay result with imipenem was obtained with a purified enzymatic extract of this transformant strain. Therefore, we consider this new beta-lactamase to be involved in the carbapenem resistance of A. baumannii RYC 52763/97.

FIG. 1

Nucleotide sequence of the bla_OXA-24 gene. The boldface atg and taa represent the initiation and termination codons, respectively. The deduced amino acid sequence of OXA-24 is shown below the nucleotide sequence. Amino acids of the signal peptide, written in lowercase and bold letters, were identified using the Swiss-Prot annotated protein sequence database (http://www.expasy.ch/sprot/sprot-top.html/). The β-lactamase active site STFK, the typical motif FGN, and the triad KSG are indicated by bold letters. The locations of the primers used to sequence the gene are underlined.

FIG. 2

Phylogram relating OXA-24 to 12 other class D β-lactamases. Analysis was performed using the CLUSTAL W multiple sequence alignment program. A gap penalty of three was applied, and 100 bootstrap replications were applied.

FIG. 3

Microbiological assay plate showing inactivation of imipenem (central disk) by the A. baumannii OXA-24 enzyme. (1) Twenty microliters of semipurified OXA-24 protein (nitrocefin-specific activity, 251.5 μmol/min/μl); (2) 10 μl of the same extract; (3) 5 μl of the same extract; (4) 20 μl of phosphate-buffered saline.