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OXA-11, an extended-spectrum variant of OXA-10 (PSE-2) beta-lactamase from Pseudomonas aeruginosa
L M Hall1, D M Livermore, D Gur
1Department of Medical Microbiology, London Hospital Medical College, United Kingdom.
Abstract:
Pseudomonas aeruginosa ABD, which was isolated in October 1991 from blood cultures of a burn patient in Turkey, was resistant to cephalosporins, particularly ceftazidime (MIC, 512 micrograms/ml), penicillins, aztreonam, and meropenem, but not to imipenem. Cephalosporin and penicillin resistance transferred to P. aeruginosa PU21 and was associated with a beta-lactamase with a pI of 6.4 encoded by a 100-MDa plasmid designated pMLH52. Like extended-spectrum TEM and SHV beta-lactamases, this enzyme hydrolyzed penicillins and newer cephalosporins but did not hydrolyze cefoxitin or carbapenems. However, it differed from TEM and SHV derivatives in being a potent oxacillinase, and its encoding gene did not hybridize with probes to TEM and SHV genes. To characterize the enzyme, libraries of total DNA were cloned into plasmid pUC19 and were transformed into Escherichia coli DH5 alpha. Recombinant plasmids that gave ceftazidime resistance all contained a 3.65-kb BamHI fragment. Deletions from this fragment allowed the beta-lactamase gene to be located on a 1.4-kb section of DNA, which contained an open reading frame of 798 bases. This encoded a protein that was deduced to differ from PSE-2 beta-lactamase only in having serine instead of asparagine at position 143 and aspartate instead of glycine at position 157. It is concluded that the resistance of isolate ABD dependent on an extended-spectrum variant of the PSE-2 enzyme. The ability of this enzyme to cause ceftazidime resistance dependent primarily on a low Km for the compound; Vmax remained low. It is proposed that PSE-2 should be transferred to the OXA group as OXA-10 and that the new enzyme be designated OXA-11.
Insights
A novel beta-lactamase, OXA-11, was identified in Pseudomonas aeruginosa, conferring resistance to ceftazidime and other cephalosporins. This extended-spectrum enzyme, a variant of PSE-2, highlights evolving antibiotic resistance mechanisms.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Antibiotic resistance in Pseudomonas aeruginosa poses a significant clinical challenge.
- Extended-spectrum beta-lactamases (ESBLs) are a major mechanism of resistance to cephalosporins.
- The emergence of novel beta-lactamases necessitates continuous surveillance and characterization.
Purpose of the Study:
- To identify and characterize the beta-lactamase responsible for ceftazidime resistance in Pseudomonas aeruginosa isolate ABD.
- To determine the genetic basis and biochemical properties of the novel beta-lactamase.
- To propose a new classification for the identified enzyme.
Main Methods:
- Isolation and characterization of Pseudomonas aeruginosa ABD from a burn patient.
- Plasmid-mediated transfer of resistance to susceptible strains.
- Cloning of DNA libraries into Escherichia coli for functional screening.
- DNA sequencing and protein analysis to determine enzyme structure and function.
- Biochemical assays to determine substrate hydrolysis profile and kinetic parameters.
Main Results:
- A beta-lactamase conferring resistance to ceftazidime, penicillins, and aztreonam was identified, encoded by plasmid pMLH52.
- The enzyme, a variant of PSE-2 beta-lactamase, showed potent oxacillinase activity and hydrolyzed newer cephalosporins.
- Genetic analysis revealed a 798-base open reading frame encoding the novel beta-lactamase, designated OXA-11.
- Kinetic analysis indicated that resistance to ceftazidime was primarily due to a low Km for the compound.
Conclusions:
- The ceftazidime resistance in Pseudomonas aeruginosa ABD is mediated by a novel extended-spectrum beta-lactamase, OXA-11.
- OXA-11 represents a significant development in beta-lactamase evolution, differing from known TEM and SHV enzymes.
- The proposed classification of this enzyme as OXA-11 provides a framework for understanding and tracking its spread.