Ourghanlian Clément, Soroka Daria, Arthur Michel
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.
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
Antimicrob Agents Chemother. 2017 Feb 23;61(3). doi: 10.1128/AAC.02510-16. Print 2017 Mar.
The substitution NG 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 from and 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 NG 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 NG 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 NG, 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 NG 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 NG 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.
先前研究表明，快速生长分枝杆菌的A类β-内酰胺酶的SDN基序中替换为NG会削弱阿维巴坦对其的抑制作用，但会提高与克拉维酸形成的酰基酶的稳定性。在KPC-2和CTX-M-15中引入相同的替换，以评估其对β-内酰胺酶的影响，并评估其是否可能导致对头孢他啶-阿维巴坦联合制剂产生耐药性。以硝基头孢菌素为底物，通过分光光度法测定阿维巴坦和克拉维酸对β-内酰胺酶的抑制动力学参数。NG替换削弱了（>1000倍）阿维巴坦对KPC-2和CTX-M-15的氨甲酰化作用。由于脱酰作用减少，该替换提高了克拉维酸对KPC-2的抑制作用，而NG的存在与否均导致克拉维酸对CTX-M-15的抑制作用。NG替换对KPC-2和CTX-M-15水解阿莫西林和硝基头孢菌素的影响适中，但对头孢他啶、头孢洛林和氨曲南的水解作用则大幅降低。构建了产生KPC-2和CTX-M-15的同基因菌株，以评估NG替换对β-内酰胺酶抑制剂联合制剂抗菌活性的影响。对于阿莫西林，该替换分别导致对阿维巴坦耐药和对克拉维酸敏感。对于头孢他啶、头孢洛林和氨曲南，该替换对β-内酰胺酶活性的负面影响阻止了对β-内酰胺-阿维巴坦联合制剂产生耐药性。总之，NG替换对A类β-内酰胺酶的底物和抑制谱有深远影响，这些酶在远缘相关酶中基本保守。幸运的是，该替换不会导致对头孢他啶-阿维巴坦联合制剂产生耐药性。
