title: Inhibition of β-lactamases of mycobacteria by avibactam and clavulanate.
abstract: Mycobacterium tuberculosis and Mycobacterium abscessus produce broad-spectrum class A β-lactamases, BlaC and Bla Mab , which are inhibited by clavulanate and avibactam, respectively. BlaC differs from Bla Mab at Ambler position 132 in the conserved motif SDN (SDG versus SDN, respectively). Here, we investigated whether this polymorphism could account for the inhibition specificity of β-lactamases from slowly and rapidly growing mycobacteria.
  
  
  
  Enzyme kinetics were determined to assess the impact of the substitutions G 132 N in BlaC and N 132 G in Bla Mab on β-lactamase inhibition by clavulanate and avibactam. The stability of acylenzymes was evaluated by MS. The impact of the substitutions on the antibacterial activity of drug combinations was determined based on production of the β-lactamases in Escherichia coli .
  
  
  
  The substitution G 132 N increased 140-fold the efficacy of BlaC inhibition by avibactam and abolished clavulanate inhibition due to acylenzyme hydrolysis. Bla Mab efficiently hydrolysed clavulanate, but the substitution N 132 G led to a 5600-fold reduction in the hydrolysis rate constant k cat due to stabilization of Bla Mab -clavulanate covalent adducts. The N 132 G substitution also led to a 610-fold reduction in the efficacy of Bla Mab carbamylation by avibactam. Testing resistance to the amoxicillin/clavulanate and amoxicillin/avibactam combinations revealed that modifications in the catalytic properties of the β-lactamases resulted in opposite shifts from susceptibility to resistance and vice versa.
  
  
  
  G 132 N and N 132 G had opposite effects on the inhibition of BlaC and Bla Mab , indicating that these substitutions might lead to acquisition of resistance to either of the β-lactamase inhibitors, but not to both of them.
authors.affiliation: INSERM, U1138, LRMA, Equipe 12 du Centre de Recherche des Cordeliers, 75006 Paris, France.|Université Pierre et Marie Curie, Paris, France UMR S 1138.|Sorbonne Paris Cité, Université Paris Descartes, UMR S 1138, Paris, France.
authors.forename: Daria
authors.initials: D
authors.lastname: Soroka
authors.affiliation: INSERM, U1138, LRMA, Equipe 12 du Centre de Recherche des Cordeliers, 75006 Paris, France.|Université Pierre et Marie Curie, Paris, France UMR S 1138.|Sorbonne Paris Cité, Université Paris Descartes, UMR S 1138, Paris, France.
authors.forename: Clément
authors.initials: C
authors.lastname: Ourghanlian
authors.affiliation: INSERM, U1138, LRMA, Equipe 12 du Centre de Recherche des Cordeliers, 75006 Paris, France.|Université Pierre et Marie Curie, Paris, France UMR S 1138.|Sorbonne Paris Cité, Université Paris Descartes, UMR S 1138, Paris, France.|Assistance Publique-Hôpitaux de Paris, Service de Microbiologie, Hôpital Européen Georges Pompidou, Paris, France.
authors.forename: Fabrice
authors.initials: F
authors.lastname: Compain
authors.affiliation: INSERM, U1138, LRMA, Equipe 12 du Centre de Recherche des Cordeliers, 75006 Paris, France.|Université Pierre et Marie Curie, Paris, France UMR S 1138.|Sorbonne Paris Cité, Université Paris Descartes, UMR S 1138, Paris, France.
authors.forename: Marion
authors.initials: M
authors.lastname: Fichini
authors.affiliation: INSERM, U1138, LRMA, Equipe 12 du Centre de Recherche des Cordeliers, 75006 Paris, France.|Université Pierre et Marie Curie, Paris, France UMR S 1138.|Sorbonne Paris Cité, Université Paris Descartes, UMR S 1138, Paris, France.|Assistance Publique-Hôpitaux de Paris, Service de Réanimation Médicale, Hôpital Saint Antoine, Paris, France.
authors.forename: Vincent
authors.initials: V
authors.lastname: Dubée
authors.affiliation: INSERM, U1138, LRMA, Equipe 12 du Centre de Recherche des Cordeliers, 75006 Paris, France.|Université Pierre et Marie Curie, Paris, France UMR S 1138.|Sorbonne Paris Cité, Université Paris Descartes, UMR S 1138, Paris, France.|Assistance Publique-Hôpitaux de Paris, Service de Microbiologie, Hôpital Européen Georges Pompidou, Paris, France.
authors.forename: Jean-Luc
authors.initials: JL
authors.lastname: Mainardi
authors.affiliation: INSERM, U1138, LRMA, Equipe 12 du Centre de Recherche des Cordeliers, 75006 Paris, France.|Université Pierre et Marie Curie, Paris, France UMR S 1138.|Sorbonne Paris Cité, Université Paris Descartes, UMR S 1138, Paris, France.
authors.forename: Jean-Emmanuel
authors.initials: JE
authors.lastname: Hugonnet
authors.affiliation: INSERM, U1138, LRMA, Equipe 12 du Centre de Recherche des Cordeliers, 75006 Paris, France.|Université Pierre et Marie Curie, Paris, France UMR S 1138.|Sorbonne Paris Cité, Université Paris Descartes, UMR S 1138, Paris, France.
authors.forename: Michel
authors.initials: M
authors.lastname: Arthur
mesh_terms: D019943:Amino Acid Substitution; D053961:Azabicyclo Compounds / Q000378:metabolism*; D019818:Clavulanic Acid / Q000378:metabolism*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D013058:Mass Spectrometry; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D009161:Mycobacterium / Q000201:enzymology*; D065093:beta-Lactamase Inhibitors / Q000378:metabolism*; D001618:beta-Lactamases / Q000378:metabolism*
chemical_list: D053961:Azabicyclo Compounds; D050505:Mutant Proteins; D065093:beta-Lactamase Inhibitors; D019818:Clavulanic Acid; C543519:avibactam; D001618:beta-Lactamases