Journal Article

Characterization of a novel Zn2+-dependent intrinsic imipenemase from Pseudomonas aeruginosa

Alicia Fajardo, Sara Hernando-Amado, Antonio Oliver, Geneviève Ball, Alain Filloux and Jose L. Martinez

in Journal of Antimicrobial Chemotherapy

Volume 69, issue 11, pages 2972-2978
Published in print November 2014 | ISSN: 0305-7453
Published online July 2014 | e-ISSN: 1460-2091 | DOI: https://dx.doi.org/10.1093/jac/dku267
Characterization of a novel Zn2+-dependent intrinsic imipenemase from Pseudomonas aeruginosa

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Objectives

Previous work showed that PA5542 inactivation increases Pseudomonas aeruginosa 59.20 susceptibility to carbapenems. The objective of the current study was to purify PA5542, to determine its role in carbapenem resistance and to analyse the kinetic constants of this putative new β-lactamase.

Methods

PA5542 was cloned and expressed in Escherichia coli. The enzyme was purified by affinity as a GST fusion protein and, after that, cleaved to remove the GST tag. β-Lactamase activity was measured spectrophotometrically using imipenem as substrate. Susceptibility to antibiotics was determined by Etest. Zn2+ was added when needed. The expression levels of PA5542, ampC, poxB, mexA and oprD were determined by real-time RT–PCR.

Results

Lack of PA5542 increases P. aeruginosa 59.20 susceptibility to carbapenems and its overexpression reduces E. coli susceptibility to these β-lactams. PA5542 is highly conserved in all sequenced P. aeruginosa strains. The clinical isolate 59.20 is resistant to imipenem (MIC >32 mg/L) and to meropenem (MIC 24 mg/L) and presents high-level expression of PA5542 in comparison with the wild-type strain PAO1. Spectrophotometric analyses showed that PA5542 is a Zn2+-dependent imipenemase. Analysis of the PA5542 sequence indicates that it does not belong to the classical categories of β-lactamases.

Conclusions

PA5542 encodes a new Zn2+-dependent imipenemase. The presence of PA5542 in all sequenced P. aeruginosa genomes, maintaining the synteny and without adjacent gene-mobility elements, indicates that it belongs to the P. aeruginosa core genome. High PA5542 expression in 59.20 suggests it may contribute to the resistance to carbapenems of this P. aeruginosa clinical isolate.

Keywords: metallo-β-lactamases; carbapenemases; intrinsic resistance; resistome

Journal Article.  4080 words.  Illustrated.

Subjects: Medical Oncology ; Critical Care

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