Blazquez J, Morosini M I, Negri M C, Gonzalez-Leiza M, Baquero F
Servicio de Microbiología, Hospital Ramón y Cajal, Madrid, Spain.
Antimicrob Agents Chemother. 1995 Jan;39(1):145-9. doi: 10.1128/AAC.39.1.145.
By directed mutagenesis, we constructed a set of seven TEM-1 derivatives containing single replacements in each one of the amino acids substituted in naturally occurring extended-spectrum TEM beta-lactamases. The exact contribution of each mutation to the resistance phenotype was determined. In addition, mutant enzyme production and stabilities were studied. Five of seven mutations determined to some extent variations in cephalosporin and/or monobactam activity. Dramatic changes in the hydrolysis of ceftazidime and aztreonam occurred when a serine was at position 164. Changes at positions 104, 238, and 240 showed more leaky variation in activity towards cephalosporins and aztreonam. Replacements at positions 237 and 265 caused no variation in susceptibility to cephalosporins. Interestingly, the change from Gln to Lys at position 39 found in TEM-2, classically considered a neutral change, slightly but consistently increased the MIC of ceftazidime and aztreonam. The in vitro construction of mutations appearing in naturally occurring TEM-beta-lactamases, studied in the same genetic context, may help to understand the evolution of extended-spectrum beta-lactamases.
通过定向诱变，我们构建了一组七个TEM-1衍生物，它们在天然存在的超广谱TEMβ-内酰胺酶中被取代的每个氨基酸处都含有单个替换。确定了每个突变对耐药表型的确切贡献。此外，还研究了突变酶的产生和稳定性。七个突变中的五个在一定程度上决定了头孢菌素和/或单环β-内酰胺活性的变化。当丝氨酸位于第164位时，头孢他啶和氨曲南的水解发生了显著变化。第104、238和240位的变化对头孢菌素和氨曲南的活性表现出更不明显的变化。第237和265位的替换对头孢菌素的敏感性没有影响。有趣的是，在TEM-2中发现的第39位从谷氨酰胺到赖氨酸的变化，传统上被认为是中性变化，但却略微但持续地增加了头孢他啶和氨曲南的最低抑菌浓度。在相同遗传背景下研究天然存在的TEM-β-内酰胺酶中出现的突变的体外构建，可能有助于理解超广谱β-内酰胺酶的进化。
