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Characterization of the First OXA-10 Natural Variant with Increased Carbapenemase Activity.

ABSTRACT: While carbapenem resistance in Gram-negative bacteria is mainly due to the production of efficient carbapenemases, ?-lactamases with a narrower spectrum may also contribute to resistance when combined with additional mechanisms. OXA-10-type class D ?-lactamases, previously shown to be weak carbapenemases, could represent such a case. In this study, two novel OXA-10 variants were identified as the sole carbapenem-hydrolyzing enzymes in meropenem-resistant enterobacteria isolated from hospital wastewater and found by next-generation sequencing to express additional ?-lactam resistance mechanisms. The new variants, OXA-655 and OXA-656, were carried by two related IncQ1 broad-host-range plasmids. Compared to the sequence of OXA-10, they both harbored a Thr26Met substitution, with OXA-655 also bearing a leucine instead of a valine in position 117 of the SAV catalytic motif. Susceptibility profiling of laboratory strains replicating the natural bla OXA plasmids and of recombinant clones expressing OXA-10 and the novel variants in an isogenic background indicated that OXA-655 is a more efficient carbapenemase. The carbapenemase activity of OXA-655 is due to the Val117Leu substitution, as shown by steady-state kinetic experiments, where the k cat of meropenem hydrolysis was increased 4-fold. In contrast, OXA-655 had no activity toward oxyimino-?-lactams, while its catalytic efficiency against oxacillin was significantly reduced. Moreover, the Val117Leu variant was more efficient against temocillin and cefoxitin. Molecular dynamics indicated that Val117Leu affects the position 117-Leu155 interaction, leading to structural shifts in the active site that may alter carbapenem alignment. The evolutionary potential of OXA-10 enzymes toward carbapenem hydrolysis combined with their spread by promiscuous plasmids indicates that they may pose a future clinical threat.

SUBMITTER: Kotsakis SD

PROVIDER: S-EPMC6325193 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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Characterization of the First OXA-10 Natural Variant with Increased Carbapenemase Activity.

Kotsakis Stathis D SD Flach Carl-Fredrik CF Razavi Mohammad M Larsson D G Joakim DGJ

Antimicrobial agents and chemotherapy 20181221 1

While carbapenem resistance in Gram-negative bacteria is mainly due to the production of efficient carbapenemases, β-lactamases with a narrower spectrum may also contribute to resistance when combined with additional mechanisms. OXA-10-type class D β-lactamases, previously shown to be weak carbapenemases, could represent such a case. In this study, two novel OXA-10 variants were identified as the sole carbapenem-hydrolyzing enzymes in meropenem-resistant enterobacteria isolated from hospital was ...[more]

PMID: 30397053

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Project description:Purpose:A novel variant of OXA-23, named OXA-423, was identified in an Acinetobacter baumannii clinical isolate. The aim of this study was to analyse the resistance phenotype of OXA-423. Methods:The A. baumannii strain WY-0713 was isolated from an intensive care unit patient. PCR was used to detect the bla OXA-23-like genes. Amplifying, cloning and sequencing were performed for the complete bla OXA-23-like. The novel bla OXA-423 and its ancestor bla OXA-23 were cloned into the expression vector pET-28b(+), and transformed into E. coli Rosetta (DE3) for antibiotic susceptibility testing. SDS-PAGE, modified Hodge test and CarbaNP test were used for detecting the expression of OXA-423 and OXA-23. Results:PCR screening of A. baumannii WY-0713 was positive for bla OXA-23-like genes. Sequencing of the PCR product identi?ed a novel bla OXA-23-like, named bla OXA-423 which encoding OXA-423. OXA-423 differed from OXA-23 by a crucial amino acid substitution (Val128Ala). The V128A substitution was located at the conserved active-site motifs SAV of OXA-23. Antibiotic susceptibility testing performed using isogenic E. coli showed that the MICs of E. coli Rosetta (pET-OXA-423) for penicillins and carbapenems were lower (reduced MICs 4-fold to 16-fold) than that of E. coli Rosetta (pET-OXA-23). The MICs of cefotaxime, ceftazidime and aztreonam for both transformants remained the same as the acceptor strain. Moreover, OXA-423 was slightly inhibited by sulbactam, clavulanic acid and tazobactam. SDS-PAGE analysis showed that OXA-423 and OXA-23 were conspicuously expressed. Modified Hodge test and CarbaNP test were positive demonstrated both of them were functional. Conclusion:OXA-423, the first report of an amino acid substitution located at conserved active-site motifs of OXA-23, conferred lower MIC values of penicillins and carbapenems as compared with OXA-23, while without affecting the resistance pro?les of expanded-spectrum cephalosporins and aztreonam.

Dataset Information

Characterization of the First OXA-10 Natural Variant with Increased Carbapenemase Activity.

Publications

Characterization of the First OXA-10 Natural Variant with Increased Carbapenemase Activity.

Similar Datasets

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