title: Characterization of a novel Zn²⁺-dependent intrinsic imipenemase from Pseudomonas aeruginosa.
abstract: OBJECTIVE
  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. Zn(2+) 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 Zn(2+)-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 Zn(2+)-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.
authors.affiliation: Departamento de Biotecnología Microbiana, Centro Nacional de Biotecnología, CSIC, Darwin 3, Cantoblanco, Madrid, Spain.
authors.forename: Alicia
authors.initials: A
authors.lastname: Fajardo
authors.affiliation: Departamento de Biotecnología Microbiana, Centro Nacional de Biotecnología, CSIC, Darwin 3, Cantoblanco, Madrid, Spain.
authors.forename: Sara
authors.initials: S
authors.lastname: Hernando-Amado
authors.affiliation: Servicio de Microbiología, Hospital Son Espases, Instituto de Investigación Sanitaria de Palma, Ctra Valldemossa 79, 07010 Palma de Mallorca, Spain.
authors.forename: Antonio
authors.initials: A
authors.lastname: Oliver
authors.affiliation: Laboratoire d'Ingénierie des Systèmes Macromoléculaires LISM-UMR7255 CNRS/Aix-Marseille Université, IMM, 31 Chemin Joseph Aiguier, 13402 Marseille cedex 20, France.
authors.forename: Geneviève
authors.initials: G
authors.lastname: Ball
authors.affiliation: Laboratoire d'Ingénierie des Systèmes Macromoléculaires LISM-UMR7255 CNRS/Aix-Marseille Université, IMM, 31 Chemin Joseph Aiguier, 13402 Marseille cedex 20, France.
authors.forename: Alain
authors.initials: A
authors.lastname: Filloux
authors.affiliation: Departamento de Biotecnología Microbiana, Centro Nacional de Biotecnología, CSIC, Darwin 3, Cantoblanco, Madrid, Spain jlmtnez@cnb.csic.es.
authors.forename: Jose L
authors.initials: JL
authors.lastname: Martinez
mesh_terms: D001426:Bacterial Proteins / Q000302:isolation & purification* / Q000378:metabolism; D015780:Carbapenems / Q000494:pharmacology; D006801:Humans; D015378:Imipenem / Q000494:pharmacology; D011550:Pseudomonas aeruginosa / Q000187:drug effects / Q000201:enzymology* / Q000302:isolation & purification*; D015032:Zinc
chemical_list: D001426:Bacterial Proteins; D015780:Carbapenems; D015378:Imipenem; D015032:Zinc
keywords: carbapenemases; intrinsic resistance; metallo-β-lactamases; resistome