title: Genetic and biochemical characterization of OXA-63, a new class D beta-lactamase from Brachyspira pilosicoli BM4442.
abstract: Brachyspira pilosicoli BM4442, isolated from the feces of a patient with diarrhea at the Hospital Saint-Michel in Paris, was resistant to oxacillin (MIC > 256 microg/ml) but remained susceptible to cephalosporins and to the combination of amoxicillin and clavulanic acid. Cloning and sequencing of the corresponding resistance determinant revealed a coding sequence of 807 bp encoding a new class D beta-lactamase named OXA-63. The bla OXA-63 gene was chromosomally located and not part of a transposon or of an integron. OXA-63 shared 54% identity with FUS-1 (OXA-85), an oxacillinase from Fusobacterium nucleatum subsp. polymorphum, and 25 to 44% identity with other class D beta-lactamases (DBLs) and contained all the conserved structural motifs of DBLs. Escherichia coli carrying the bla OXA-63 gene exhibited resistance to benzylpenicillin and amoxicillin but remained susceptible to amoxicillin in combination with clavulanic acid. Mature OXA-63 consisted of a 31.5-kDa polypeptide and appeared to be dimeric. Kinetic analysis revealed that OXA-63 exhibited a narrow substrate profile, hydrolyzing oxacillin, benzylpenicillin, and ampicillin with catalytic efficiencies of 980, 250, and 150 mM(-1) s(-1), respectively. The enzyme did not apparently interact with oxyimino-cephalosporins, imipenem, or aztreonam. Unlike FUS-1 and other DBLs, OXA-63 was strongly inhibited by clavulanic acid (50% inhibitory concentration [IC50] of 2 microM) and tazobactam (IC50 of 0.16 microM) and exhibited low susceptibility to NaCl (IC50 of >2 M). OXA-63 is the first DBL described for the anaerobic spirochete B. pilosicoli.
authors.affiliation: Unité des Agents Antibactériens, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15, France.
authors.forename: Djalal
authors.initials: D
authors.lastname: Meziane-Cherif
authors.forename: Thierry
authors.initials: T
authors.lastname: Lambert
authors.forename: Marine
authors.initials: M
authors.lastname: Dupêchez
authors.forename: Patrice
authors.initials: P
authors.lastname: Courvalin
authors.forename: Marc
authors.initials: M
authors.lastname: Galimand
mesh_terms: D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001483:Base Sequence; D017400:Brachyspira / Q000187:drug effects* / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D003001:Cloning, Molecular; D003967:Diarrhea / Q000382:microbiology; D005243:Feces / Q000382:microbiology; D016905:Gram-Negative Bacterial Infections / Q000382:microbiology; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D010068:Oxacillin / Q000494:pharmacology; D010403:Penicillin Resistance; D017422:Sequence Analysis, DNA; D001618:beta-Lactamases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D000900:Anti-Bacterial Agents; D001618:beta-Lactamases; C108449:oxacillinase; D010068:Oxacillin