title: Genetic and functional analysis of the chromosome-encoded carbapenem-hydrolyzing oxacillinase OXA-40 of Acinetobacter baumannii.
abstract: Clinical isolate Acinetobacter baumannii CLA-1 was resistant to a series of antibiotic molecules, including carbapenems. Cloning and expression of the beta-lactamase gene content of this isolate in Escherichia coli DH10B identified a chromosome-encoded oxacillinase, OXA-40, that differed by one or two amino acid changes from OXA-24, -25, and -26 and an AmpC-type cephalosporinase. The OXA-40 beta-lactamase had a mainly narrow-spectrum hydrolytic profile, but it included ceftazidime and imipenem. Its activity was resistant to inhibition by clavulanic acid, tazobactam, sulbactam, and, like most of the other carbapenem-hydrolyzing oxacillinases, NaCl. OXA-40 had an FGN triad replacing a YGN motif at class D beta-lactamase (DBL) positions 144 to 146. Site-directed DNA mutagenesis leading to a Phe-to-Tyr change at DBL position 144 in OXA-40 gave a mutant enzyme with increased hydrolytic activity against most beta-lactams, including imipenem. Conversely, with a gene encoding the narrow-spectrum oxacillinase OXA-1 as the template, a nucleotide substitution leading to a Tyr-to-Phe change in the YGN motif of OXA-1 gave a mutant enzyme with decreased hydrolytic activity without an increase in carbapenem-hydrolyzing activity. Thus, the Phe residue in the FGN motif was not associated with carbapenem-hydrolyzing activity by itself but instead was associated with weak overall hydrolytic activity. Finally, this Phe residue in OXA-40 explained resistance to inhibition by NaCl whereas a Tyr residue in motif YGN was related to susceptibility to NaCl.
authors.affiliation: Service de Bactériologie-Virologie, Hôpital de Bicêtre, Assistance Publique/Hôpitaux de Paris, Faculté de Médecine Paris-Sud, 94275 Le Kremlin-Bicêtre, France.
authors.forename: Claire
authors.initials: C
authors.lastname: Héritier
authors.forename: Laurent
authors.initials: L
authors.lastname: Poirel
authors.forename: Daniel
authors.initials: D
authors.lastname: Aubert
authors.forename: Patrice
authors.initials: P
authors.lastname: Nordmann
mesh_terms: D040981:Acinetobacter baumannii / Q000187:drug effects / Q000201:enzymology*; D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000187:drug effects / Q000235:genetics*; D004926:Escherichia coli / Q000187:drug effects; D008826:Microbial Sensitivity Tests; D016296:Mutagenesis; D001618:beta-Lactamases / Q000187:drug effects / Q000235:genetics*
chemical_list: D002352:Carrier Proteins; D001618:beta-Lactamases; C108449:oxacillinase