Stapleton P D, Shannon K P, French G L
Department of Microbiology, The Guy's, King's & St. Thomas' School of Medicine, St. Thomas' Campus, London SE1 7EH, United Kingdom.
Antimicrob Agents Chemother. 1999 Aug;43(8):1881-7. doi: 10.1128/AAC.43.8.1881.
Extended-spectrum TEM beta-lactamases (ESBLs) do not usually confer resistance to beta-lactamase inhibitors such as clavulanate or tazobactam. To investigate the compatibility of the two phenotypes we used site-directed mutagenesis of the bla(TEM-1) gene to introduce into the TEM-1 beta-lactamase amino acid substitutions that confer the ESBL phenotype: TEM-12 (Arg164-->Ser), TEM-26 (Arg164-->Ser plus Glu104-->Lys), TEM-19 (Gly238-->Ser), and TEM-15 (Gly238-->Ser plus Glu104-->Lys). These were combined with three sets of substitutions that confer inhibitor resistance: TEM-31 (Arg244-->Cys), TEM-33 (Met69-->Leu), and TEM-35 (Met69-->Leu and Asn276-->Asp). Introduction of the Arg244-->Cys substitution gave rise to inhibitor-resistant hybrid enzymes that either lost ESBL activity (TEM-12, TEM-15, and TEM-19) or had reduced activity (TEM-26) against ceftazidime. In contrast, the introduction of Met69-->Leu or Met69-->Leu plus Asn276-->Asp substitutions did not significantly affect the abilities of the enzymes to confer resistance to ceftazidime, although increased susceptibility to cefotaxime was observed with Escherichia coli strains that expressed the TEM-19 and TEM-26 beta-lactamases. With the exception of the TEM-12 beta-lactamase, introduction of the Met69-->Leu substitution did not give rise to enzymes with increased resistance to clavulanate compared to that of the TEM-1 beta-lactamase. However, introduction of the double substitution Met69-->Leu plus Asn276-->Asp in the ESBLs did give rise to low-level (TEM-19, TEM-15, and TEM-26) or moderate-level (TEM-12) clavulanate resistance. None of the hybrid enzymes were as resistant to clavulanate as the corresponding inhibitor-resistant TEM beta-lactamase mutant, suggesting that active-site configuration in the ESBLs limits the degree of clavulanate resistance conferred.
超广谱 TEM β-内酰胺酶（ESBLs）通常不会赋予对诸如克拉维酸或他唑巴坦等β-内酰胺酶抑制剂的耐药性。为了研究这两种表型的兼容性，我们使用 bla(TEM-1) 基因的定点诱变，将赋予 ESBL 表型的氨基酸取代引入 TEM-1 β-内酰胺酶中：TEM-12（Arg164→Ser）、TEM-26（Arg164→Ser 加 Glu104→Lys）、TEM-19（Gly238→Ser）和 TEM-15（Gly238→Ser 加 Glu104→Lys）。这些与三组赋予抑制剂耐药性的取代组合：TEM-31（Arg244→Cys）、TEM-33（Met69→Leu）和 TEM-35（Met69→Leu 加 Asn276→Asp）。引入 Arg244→Cys 取代产生了对抑制剂耐药的杂合酶，这些酶对头孢他啶要么失去 ESBL 活性（TEM-12、TEM-15 和 TEM-19），要么活性降低（TEM-26）。相比之下，引入 Met69→Leu 或 Met69→Leu 加 Asn276→Asp 取代并没有显著影响这些酶赋予对头孢他啶耐药性的能力，尽管在表达 TEM-19 和 TEM-26 β-内酰胺酶的大肠杆菌菌株中观察到对头孢噻肟的敏感性增加。除了 TEM-12 β-内酰胺酶外，与 TEM-1 β-内酰胺酶相比，引入 Met69→Leu 取代并没有产生对克拉维酸耐药性增加的酶。然而，在 ESBLs 中引入双取代 Met69→Leu 加 Asn276→Asp 确实产生了低水平（TEM-19、TEM-15 和 TEM-26）或中等水平（TEM-12）的克拉维酸耐药性。没有一种杂合酶对克拉维酸的耐药性与相应的对抑制剂耐药的 TEM β-内酰胺酶突变体一样高，这表明 ESBLs 中的活性位点构型限制了所赋予的克拉维酸耐药程度。
