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Molecular characterization of OXA-20, a novel class D beta-lactamase, and its integron from Pseudomonas aeruginosa.

ABSTRACT: The Pseudomonas aeruginosa Mus clinical isolate produces OXA-18, a pI 5.5 class D extended-spectrum beta-lactamase totally inhibited by clavulanic acid (L. N. Philippon, T. Naas, A.-T. Bouthors, V. Barakett, and P. Nordmann, Antimicrob. Agents Chemother. 41:2188-2195, 1997). A second beta-lactamase was cloned, and the recombinant Escherichia coli clone pPL10 expressed a pI 7.4 beta-lactamase which conferred high levels of amoxicillin and ticarcillin resistance and which was partially inhibited by clavulanic acid. The 2.5-kb insert from pPL10 was sequenced, and a 266-amino-acid protein (OXA-20) was deduced; this protein has low amino acid identity with most of the class D beta-lactamases except OXA-2, OXA-15, and OXA-3 (75% amino acid identity with each). OXA-20 is a restricted-spectrum oxacillinase and is unusually inhibited by clavulanic acid. OXA-20 is a peculiar beta-lactamase because its translation initiates with a TTG (leucine) codon, which is rarely used as a translational origin in bacteria. Exploration of the genetic environment of oxa20 revealed the presence of the following integron features: (i) a second antibiotic resistance gene, aacA4; (ii) an intI1 gene; and (iii) two 59-base elements, each associated with either oxa20 or aacA4. This integron is peculiar because it lacks the 3' conserved region, and therefore is not a sul1-associated integron like most of them, and because its 3' end is located within tnpR, a gene involved in the transposition of Tn5393, a gram-negative transposon. P. aeruginosa Mus produces two novel and unrelated oxacillinases, OXA-18 and OXA-20, both of which are inhibited by clavulanic acid.

SUBMITTER: Naas T

PROVIDER: S-EPMC105865 | biostudies-literature | 1998 Aug

REPOSITORIES: biostudies-literature

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Molecular characterization of OXA-20, a novel class D beta-lactamase, and its integron from Pseudomonas aeruginosa.

Naas T T Sougakoff W W Casetta A A Nordmann P P

Antimicrobial agents and chemotherapy 19980801 8

The Pseudomonas aeruginosa Mus clinical isolate produces OXA-18, a pI 5.5 class D extended-spectrum beta-lactamase totally inhibited by clavulanic acid (L. N. Philippon, T. Naas, A.-T. Bouthors, V. Barakett, and P. Nordmann, Antimicrob. Agents Chemother. 41:2188-2195, 1997). A second beta-lactamase was cloned, and the recombinant Escherichia coli clone pPL10 expressed a pI 7.4 beta-lactamase which conferred high levels of amoxicillin and ticarcillin resistance and which was partially inhibited b ...[more]

PMID: 9687410

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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

Molecular characterization of OXA-20, a novel class D beta-lactamase, and its integron from Pseudomonas aeruginosa.

Publications

Molecular characterization of OXA-20, a novel class D beta-lactamase, and its integron from Pseudomonas aeruginosa.

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