title: Inhibition by Avibactam and Clavulanate of the β-Lactamases KPC-2 and CTX-M-15 Harboring the Substitution N132G in the Conserved SDN Motif.
abstract: The substitution N132G in the SDN motif of class A β-lactamases from rapidly growing mycobacteria was previously shown to impair their inhibition by avibactam but to improve the stability of acyl-enzymes formed with clavulanate. The same substitution was introduced in KPC-2 and CTX-M-15 to assess its impact on β-lactamases fromEnterobacteriaceaeand evaluate whether it may lead to resistance to the ceftazidime-avibactam combination. Kinetic parameters for the inhibition of the β-lactamases by avibactam and clavulanate were determined by spectrophotometry using nitrocefin as the substrate. The substitution N132G impaired (>1,000-fold) the efficacy of carbamylation of KPC-2 and CTX-M-15 by avibactam. The substitution improved the inhibition of KPC-2 by clavulanate due to reduced deacylation, whereas the presence or absence of N132G resulted in the inhibition of CTX-M-15 by clavulanate. The hydrolysis of amoxicillin and nitrocefin by KPC-2 and CTX-M-15 was moderately affected by the substitution N132G, but that of ceftazidime, ceftaroline, and aztreonam was drastically reduced. Isogenic strains producing KPC-2 and CTX-M-15 were constructed to assess the impact of the substitution N132G on the antibacterial activities of β-lactam-inhibitor combinations. For amoxicillin, the substitution resulted in resistance and susceptibility for avibactam and clavulanate, respectively. For ceftazidime, ceftaroline, and aztreonam, the negative impact of the substitution on β-lactamase activity prevented resistance to the β-lactam-avibactam combinations. In conclusion, the N132G substitution has profound effects on the substrate and inhibition profiles of class A β-lactamases, which are largely conserved in distantly related enzymes. Fortunately, the substitution does not lead to resistance to the ceftazidime-avibactam combination.
authors.affiliation: INSERM, U1138, LRMA, Equipe 12 du Centre de Recherche des Cordeliers, Paris, France, Université Pierre et Marie Curie, UMR S 1138, Paris, France, and Université Paris Descartes, Sorbonne Paris Cité, 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, Paris, France, Université Pierre et Marie Curie, UMR S 1138, Paris, France, and Université Paris Descartes, Sorbonne Paris Cité, 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, Paris, France, Université Pierre et Marie Curie, UMR S 1138, Paris, France, and Université Paris Descartes, Sorbonne Paris Cité, UMR S 1138, Paris, France michel.arthur@crc.jussieu.fr.
authors.forename: Michel
authors.initials: M
authors.lastname: Arthur
mesh_terms: D020816:Amino Acid Motifs; D019943:Amino Acid Substitution; D000658:Amoxicillin / Q000494:pharmacology; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D053961:Azabicyclo Compounds / Q000494:pharmacology*; D001398:Aztreonam / Q000494:pharmacology; D002442:Ceftazidime / Q000494:pharmacology; D002511:Cephalosporins / Q000737:chemistry / Q000494:pharmacology; D019818:Clavulanic Acid / Q000494:pharmacology*; D003001:Cloning, Molecular; D004338:Drug Combinations; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D007700:Kinetics; D009154:Mutation; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D059386:Reverse Genetics; D013379:Substrate Specificity; D001618:beta-Lactamases / Q000235:genetics* / Q000378:metabolism; D000097583:Ceftaroline
chemical_list: D000658:Amoxicillin; D000900:Anti-Bacterial Agents; D053961:Azabicyclo Compounds; D001398:Aztreonam; D002442:Ceftazidime; D002511:Cephalosporins; D019818:Clavulanic Acid; D004338:Drug Combinations; D011994:Recombinant Proteins; D001618:beta-Lactamases; D000097583:Ceftaroline; C000595613:avibactam, ceftazidime drug combination; C543519:avibactam; C473109:beta-lactamase CTX-M-15; C491478:beta-lactamase KPC-2; C021720:nitrocefin
keywords: CTX-M-15; KPC-2; avibactam; clavulanate; β-lactamase inhibitor