title: Characterization of broad-spectrum Mycobacterium abscessus class A β-lactamase.
abstract: OBJECTIVE
  Imipenem and cefoxitin are used to treat Mycobacterium abscessus infections and have moderate activity against this fast-growing mycobacterium (MIC₅₀ of 16 and 32 mg/L, respectively). M. abscessus is highly resistant to most other β-lactams, although the underlying mechanisms have not been explored. Here, we characterized M. abscessus class A β-lactamase (Bla(Mab)) and investigated its role in β-lactam resistance.
  
  
  METHODS
  Hydrolysis kinetic parameters of purified Bla(Mab) were determined by spectrophotometry for various β-lactams and compared with those of related BlaC from Mycobacterium tuberculosis. MICs of β-lactams were determined for M. abscessus CIP104536 and for Escherichia coli producing Bla(Mab) and BlaC.
  
  
  RESULTS
  Bla(Mab) had a broad hydrolysis spectrum, similar to that of BlaC, but with overall higher catalytic efficiencies, except for cefoxitin. As expected from its in vivo efficacy, cefoxitin was very slowly hydrolysed by Bla(Mab) (k(cat)/K(m) = 6.7 M(-1) s(-1)). Bla(Mab) hydrolysed imipenem more efficiently (k(cat)/K(m) = 3.0 × 10(4) M(-1) s(-1)), indicating that the in vivo activity of this drug might be improved by combination with a β-lactamase inhibitor. β-Lactamase inhibitors clavulanate, tazobactam and sulbactam did not inhibit Bla(Mab). This enzyme efficiently hydrolysed clavulanate, in contrast to BlaC, which is irreversibly acylated by this inhibitor. Bla(Mab) and BlaC were functional in E. coli and the resistance profiles mediated by these enzymes were in agreement with the kinetic parameters.
  
  
  CONCLUSIONS
  M. abscessus produces a clavulanate-insensitive broad-spectrum β-lactamase that limits the in vivo efficacy of β-lactams.
authors.affiliation: INSERM, U872, LRMA, Equipe 12 du Centre de Recherche des Cordeliers, Paris F-75006, France.
authors.forename: Daria
authors.initials: D
authors.lastname: Soroka
authors.forename: Vincent
authors.initials: V
authors.lastname: Dubée
authors.forename: Olivia
authors.initials: O
authors.lastname: Soulier-Escrihuela
authors.forename: Guillaume
authors.initials: G
authors.lastname: Cuinet
authors.forename: Jean-Emmanuel
authors.initials: JE
authors.lastname: Hugonnet
authors.forename: Laurent
authors.initials: L
authors.lastname: Gutmann
authors.forename: Jean-Luc
authors.initials: JL
authors.lastname: Mainardi
authors.forename: Michel
authors.initials: M
authors.lastname: Arthur
mesh_terms: D006868:Hydrolysis; D007700:Kinetics; D009161:Mycobacterium / Q000201:enzymology*; D013379:Substrate Specificity; D018440:beta-Lactam Resistance; D001618:beta-Lactamases / Q000378:metabolism*
chemical_list: D001618:beta-Lactamases
keywords: Mycobacterium tuberculosis; cefoxitin; clavulanic acid; β-lactams