title: Ceftazidime-hydrolysing β-lactamase OXA-145 with impaired hydrolysis of penicillins in Pseudomonas aeruginosa.
abstract: OBJECTIVE
  To describe a novel extended-spectrum oxacillinase, named OXA-145, differing from narrow-spectrum OXA-35 (from the OXA-10 group) by deletion of residue Leu-165. The genetic environment of bla(OXA-145) and the biochemical properties of OXA-145 are reported. We also assessed the impact of the Leu-165 deletion on the hydrolysis spectrum of the ancestor OXA-10.
  
  
  METHODS
  Extended-spectrum β-lactamase OXA-145 was identified in the multidrug-resistant clinical Pseudomonas aeruginosa 08-056, and characterized by isoelectric focusing, PCR and DNA sequencing. Antibiotic susceptibility tests were performed by agar dilution. The resistance profiles conferred by cloned bla(OXA-10), bla(OXA-35), bla(OXA-145) and a bla(OXA-10) derivative obtained by site-directed mutagenesis were determined in Escherichia coli. Kinetic parameters of OXA-35 and OXA-145 were established after purification of His-tagged proteins.
  
  
  RESULTS
  The sequence of OXA-145, encoded by a class 1 integron-borne gene in strain 08-056, differed from that of narrow-spectrum penicillinase OXA-35 by a single amino acid deletion (Leu-165) located in the highly conserved omega loop. Deletion of Leu-165 from OXA-35 (yielding OXA-145) or OXA-10 (the progenitor of OXA-35) extended the hydrolysis spectrum to third-generation cephalosporins and to monobactams, while reducing that for penicillins. OXA-145 showed biphasic hydrolysis curves for all the substrates tested. Its activity against nitrocefin was 10-fold higher in the presence of sodium hydrogen carbonate.
  
  
  CONCLUSIONS
  OXA-145 is a new extended-spectrum β-lactamase from the OXA-10 group. The deletion of Leu-165 is responsible for a shift in the hydrolysis spectrum from penicillins to third-generation cephalosporins, as well as monobactams. The loss of penicillin hydrolysis was due to a non-carboxylated Lys-73.
authors.affiliation: Centre National de Référence de Résistance aux Antibiotiques, Laboratoire associé Pseudomonas aeruginosa, Centre Hospitalier Universitaire, Besançon, France.
authors.forename: Didier
authors.initials: D
authors.lastname: Hocquet
authors.forename: Mélanie
authors.initials: M
authors.lastname: Colomb
authors.forename: Barbara
authors.initials: B
authors.lastname: Dehecq
authors.forename: Olivier
authors.initials: O
authors.lastname: Belmonte
authors.forename: Patrice
authors.initials: P
authors.lastname: Courvalin
authors.forename: Patrick
authors.initials: P
authors.lastname: Plésiat
authors.forename: Djalal
authors.initials: D
authors.lastname: Meziane-Cherif
mesh_terms: D000900:Anti-Bacterial Agents / Q000378:metabolism*; D002442:Ceftazidime / Q000378:metabolism*; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D006868:Hydrolysis; D007526:Isoelectric Point; D007700:Kinetics; D008826:Microbial Sensitivity Tests; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D010406:Penicillins / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D017422:Sequence Analysis, DNA; D017384:Sequence Deletion; D001618:beta-Lactamases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D000900:Anti-Bacterial Agents; D004269:DNA, Bacterial; D050505:Mutant Proteins; D010406:Penicillins; D002442:Ceftazidime; D001618:beta-Lactamases