Vakulenko S B, Geryk B, Kotra L P, Mobashery S, Lerner S A
Department of Medicine, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.
Antimicrob Agents Chemother. 1998 Jul;42(7):1542-8. doi: 10.1128/AAC.42.7.1542.
Mechanism-based inactivators of beta-lactamases are used to overcome the resistance of clinical pathogens to beta-lactam antibiotics. This strategy can itself be overcome by mutations of the beta-lactamase that compromise the effectiveness of their inactivation. We used PCR mutagenesis of the TEM-1 beta-lactamase gene and sequenced the genes of 20 mutants that grew in the presence of ampicillin-clavulanate. Eleven different mutant genes from these strains contained from 1 to 10 mutations. Each had a replacement of one of the four residues, Met69, Ser130, Arg244, and Asn276, whose substitutions by themselves had been shown to result in inhibitor resistance. None of the mutant enzymes with multiple amino acid substitutions generated in this study conferred higher levels of resistance to ampicillin alone or ampicillin with beta-lactamase inactivators (clavulanate, sulbactam, or tazobactam) than the levels of resistance conferred by the corresponding single-mutant enzymes. Of the four enzymes with just a single mutation (Ser130Gly, Arg244Cys, Arg244Ser, or Asn276Asp), the Asn276Asp beta-lactamase conferred a wild-type level of ampicillin resistance and the highest levels of resistance to ampicillin in the presence of inhibitors. Site-directed random mutagenesis of the Ser130 codon yielded no other mutant with replacement of Ser130 besides Ser130Gly that produced ampicillin-clavulanate resistance. Thus, despite PCR mutagenesis we found no new mutant TEM beta-lactamase that conferred a level of resistance to ampicillin plus inactivators greater than that produced by the single-mutation enzymes that have already been reported in clinical isolates. Although this is reassuring, one must caution that other combinations of multiple mutations might still produce unexpected resistance.
基于机制的β-内酰胺酶灭活剂被用于克服临床病原体对β-内酰胺类抗生素的耐药性。这一策略本身可能会因β-内酰胺酶的突变而失效，这些突变会损害其灭活效果。我们使用PCR诱变TEM-1β-内酰胺酶基因，并对在氨苄西林-克拉维酸存在下生长的20个突变体的基因进行测序。这些菌株中的11个不同突变基因含有1至10个突变。每个突变基因都替换了四个残基（Met69、Ser130、Arg244和Asn276）中的一个，单独替换这些残基已被证明会导致抑制剂耐药。在本研究中产生的具有多个氨基酸替换的突变酶，单独对氨苄西林或与β-内酰胺酶灭活剂（克拉维酸、舒巴坦或他唑巴坦）联合使用时，其对氨苄西林的耐药水平均不高于相应单突变酶所赋予的耐药水平。在仅具有单个突变的四种酶（Ser130Gly、Arg244Cys、Arg244Ser或Asn276Asp）中，Asn276Aspβ-内酰胺酶赋予野生型水平的氨苄西林耐药性，并且在存在抑制剂的情况下对氨苄西林的耐药水平最高。对Ser130密码子进行定点随机诱变，除了产生氨苄西林-克拉维酸耐药性的Ser130Gly外，未产生其他替换Ser130的突变体。因此，尽管进行了PCR诱变，但我们未发现新的突变型TEMβ-内酰胺酶，其对氨苄西林加灭活剂的耐药水平高于临床分离株中已报道的单突变酶所产生的耐药水平。尽管这令人放心，但必须提醒的是，其他多种突变组合仍可能产生意想不到的耐药性。
