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OXA-28, an extended-spectrum variant of OXA-10 beta-lactamase from Pseudomonas aeruginosa and its plasmid- and integron-located gene.

ABSTRACT: Pseudomonas aeruginosa ED-1, isolated from a pulmonary brush of a patient hospitalized in a suburb of Paris, France, was resistant to ceftazidime and of intermediate susceptibility to ureidopenicillins and to cefotaxime. Cloning and expression of the beta-lactamase gene content of this isolate in Escherichia coli DH10B identified a novel OXA-10 variant, OXA-28, with a pI value of 8.1 and a molecular mass of 29 kDa. It differed from OXA-10 by 10 amino acid changes and from OXA-13 and OXA-19 by 2 amino acid changes, including a glycine instead of tryptophan at position 164, which is likely involved in its resistance to ceftazidime. Like OXA-11, -14, -16, and -19 and as opposed to OXA-17, OXA-28 predominantly compromised ceftazidime and had only marginal effect on the MICs of aztreonam and cefotaxime in P. aeruginosa. Once expressed in E. coli, OXA-28 raised the MIC of ceftazidime to a much higher level than those of amoxicillin, cephalothin, and cefotaxime (128, 16, 8, and 4 microg/ml, respectively). OXA-28 beta-lactamase had a broad spectrum of activity, including ceftazidime. Its activity was partially antagonized by clavulanic acid (50% inhibitory concentration, 10 microM) and NaCl addition. The oxa28 gene cassette was inserted in the variable region of a class 1 integron, In57, immediately downstream of an amino 6'-N-acetyltransferase gene cassette, aac(6')Ib. The structures of the integrons carrying either oxa28, oxa13, or oxa19 gene cassettes were almost identical, suggesting that they may have derived from a common ancestor as a result of the common European origin of the P. aeruginosa isolates. In57 was located on a self-transferable plasmid of ca. 150 kb that was transferred from P. aeruginosa to P. aeruginosa.

SUBMITTER: Poirel L

PROVIDER: S-EPMC90311 | biostudies-literature | 2001 Feb

REPOSITORIES: biostudies-literature

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OXA-28, an extended-spectrum variant of OXA-10 beta-lactamase from Pseudomonas aeruginosa and its plasmid- and integron-located gene.

Poirel L L Girlich D D Naas T T Nordmann P P

Antimicrobial agents and chemotherapy 20010201 2

Pseudomonas aeruginosa ED-1, isolated from a pulmonary brush of a patient hospitalized in a suburb of Paris, France, was resistant to ceftazidime and of intermediate susceptibility to ureidopenicillins and to cefotaxime. Cloning and expression of the beta-lactamase gene content of this isolate in Escherichia coli DH10B identified a novel OXA-10 variant, OXA-28, with a pI value of 8.1 and a molecular mass of 29 kDa. It differed from OXA-10 by 10 amino acid changes and from OXA-13 and OXA-19 by 2 ...[more]

PMID: 11158739

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Project description:Abstract Background Recently, we described a collection of ST298 Pseudomonas aeruginosa (PA) isolates that caused a prolonged epidemic of XDR infections. Many of these contain derivatives of a new plasmid, pPABL048, that harbors an MDR integron, in1697. In1697 contains a series of antimicrobial resistance (AMR) genes, one of which is the class D β-lactamase blaOXA-10. Variants of blaOXA-10 have been described that confer both extended-spectrum β-lactamase (ESBL) and carbapenemase activity. Methods Of all ST298 isolates, three were resistant to ceftazidime (CTZ). Genomic comparison of in1697 in CTZ-resistant and CTZ-sensitive strains revealed that all three strains harbored a blaOXA-10 allele with two single nucleotide variations resulting in amino acid changes at positions 153 (F153S) and 157 (G157D). Using the NCBI database, we identified this allele as unique and defined this β-lactamase as OXA-935. OXA-935 shares the G157D variation with OXA-14 which is known to confer resistance to ceftazidime. We sought to characterize the function of OXA-935 and to determine the crystal structures of OXA-14 and OXA-935. Results Deletion of blaOXA-935 phenotypically converted all three strains to CTZ-susceptible. Expression of blaOXA-14 and blaOXA-935 conferred CTZ-resistance to laboratory PA strains PA01 and PA14. Determination of the crystal structures of OXA-14 (PDB code 7L5R) and OXA-935 (PDB code 7L5V) revealed that the F153S variant resulted in increased flexibility in the enzyme’s Ω loop. Conformational changes in the Ω loop likely contributed to the lack of carbamylation at lysine-70 (K70) observed in OXA-935. Carbamylation of K70 is known to be critical for enzymatic activity of class D β-lactamases. Conclusion OXA-935 is very similar to OXA-14; however, comparison revealed that the F153S variant has unique structural features and is functionally distinct. Despite these differences, both enzymes confer high-level CTZ resistance. As we increasingly rely on β-lactam antimicrobial therapy (e.g. ceftazidime, cefepime) and combination (e.g. ceftazidime-avibactam) therapy to treat MDR PA infections, it is critical that we continue to explore the mechanistic basis of β-lactam AMR in an effort to preserve existing treatments and design novel ones. Disclosures All Authors: No reported disclosures

Dataset Information

OXA-28, an extended-spectrum variant of OXA-10 beta-lactamase from Pseudomonas aeruginosa and its plasmid- and integron-located gene.

Publications

OXA-28, an extended-spectrum variant of OXA-10 beta-lactamase from Pseudomonas aeruginosa and its plasmid- and integron-located gene.

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