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OXA-18, a class D clavulanic acid-inhibited extended-spectrum beta-lactamase from Pseudomonas aeruginosa
L N Philippon1, T Naas, A T Bouthors
1Service de Bactériologie-Virologie, Hôpital Antoine Béclère, Faculté de Médecine Paris-Sud, Clamart, France.
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 beta-lactamase. Isoelectric focusing revealed that this strain produced three beta-lactamases, of pI 5.5, 7.4, and 8.2. A 2.6-kb Sau3A fragment encoding the extended-spectrum beta-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 microg/ml. Hydrolysis constants of OXA-18 (Vmax, Km) confirmed the MIC results. Cloxacillin and oxacillin hydrolysis was noticeable with the partially purified OXA-18. The blaOXA-18 gene encodes a 275-amino-acid protein which has weak identity with all class D beta-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 beta-lactamase which confers high resistance to extended-spectrum cephalosporins and seems to have unique hydrolytic properties among non-class A enzymes.
Insights
Pseudomonas aeruginosa Mus produces OXA-18, a novel class D beta-lactamase conferring resistance to extended-spectrum cephalosporins and aztreonam. This enzyme exhibits unique hydrolytic properties and is likely chromosomally encoded.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Clinical isolates of Pseudomonas aeruginosa can exhibit resistance to crucial antibiotics like extended-spectrum cephalosporins and aztreonam.
- The presence of extended-spectrum beta-lactamases (ESBLs) is a common mechanism for such resistance.
- Characterizing novel beta-lactamases is vital for understanding and combating antimicrobial resistance.
Purpose of the Study:
- To identify and characterize the beta-lactamase responsible for resistance in Pseudomonas aeruginosa Mus.
- To determine the substrate profile and inhibitory characteristics of the novel enzyme.
- To investigate the genetic basis and potential for horizontal gene transfer of the resistance mechanism.
Main Methods:
- Double-disk synergy testing to detect ESBL production.
- Isoelectric focusing to identify beta-lactamase isoelectric points (pI).
- Cloning of the beta-lactamase gene (blaOXA-18) from genomic DNA.
- Enzyme purification and characterization of substrate hydrolysis and inhibition.
- Gene sequencing and comparison with known beta-lactamase families.
- Plasmid analysis and bacterial conjugation experiments to assess transferability.
Main Results:
- Pseudomonas aeruginosa Mus displayed resistance to extended-spectrum cephalosporins and aztreonam, with synergy observed with clavulanic acid.
- Isoelectric focusing identified three beta-lactamases with pIs of 5.5, 7.4, and 8.2.
- A 2.6-kb fragment containing the blaOXA-18 gene, encoding the pI 5.5 enzyme, was cloned and characterized.
- OXA-18, a 30.6 kDa enzyme, hydrolyzed a broad range of beta-lactams including ceftazidime and cefotaxime, but not imipenem or cephamycins, and was inhibited by clavulanic acid.
- The blaOXA-18 gene encodes a 275-amino-acid protein with weak identity to other class D beta-lactamases, except OXA-9 and OXA-12.
- No plasmids were detected, and resistance transfer failed, suggesting chromosomal encoding of OXA-18.
Conclusions:
- OXA-18 is a novel class D beta-lactamase conferring significant resistance to extended-spectrum cephalosporins and aztreonam in Pseudomonas aeruginosa.
- This enzyme possesses unique hydrolytic properties distinct from other non-class A beta-lactamases.
- The likely chromosomal location of blaOXA-18 indicates a stable resistance mechanism within this bacterial isolate.