MOLECULAR CHARACTERIZATION OF 9 DIFFERENT TYPES OF MUTANTS AMONG 107 INHIBITOR-RESISTANT TEM BETA-LACTAMASES FROM CLINICAL ISOLATES OF ESCHERICHIA-COLI
MOLECULAR CHARACTERIZATION OF 9 DIFFERENT TYPES OF MUTANTS AMONG 107 INHIBITOR-RESISTANT TEM BETA-LACTAMASES FROM CLINICAL ISOLATES OF ESCHERICHIA-COLI
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DOI：
10.1128/aac.39.2.427
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发表时间：
1995-02-01
影响因子：
4.9
通讯作者：
SIROT, J
中科院分区：
文献类型：
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作者：
HENQUELL, C;CHANAL, C;SIROT, J
对大肠杆菌临床分离株产生的107种抑制物耐药TEM （IRT）酶进行DNA-DNA杂交和测序，以确定其对克拉维酸耐药的分子基础。TEM-1酶衍生的β -内酰胺酶集中在pI 5.2 （n = 68）或5.4 (n = 39)，与TEM-1酶相比，克拉维酸对这些酶的抑制作用非常弱。结果表明，在107种酶中，有84种酶的氨基酸序列与TEM-1存在一到两个差异：22种菌株的Arg-244—>Ser (IRT-2)， 17种菌株的Met-69—>Leu (TEM-33)， 14种菌株的Met-69—>Val (TEM-34)， 6种菌株的Met-69—>Ile (IRT-3)， 13种菌株的Met-69—>Leu与Asn-276—>Asp （IRT -4）相关，12种菌株的Met-69—>Val与Asn-276—>Asp （TEM-36）相关。在20个菌株中发现了Met-69- >Ile与Asn-276- >Asp的新组合，称为IRT-8，两个以前未描述的IRT酶被表征，取代Met-69- >Val与新的取代Arg-275- >Leu发生在一个菌株中，Met-69- >Leu和Asn-276- >Asp的组合与新的取代Trp-165- >Arg发生在两个菌株中，这两个新酶分别被称为IRT-9和IRT-10。TEM-1酶的晶体学数据和定点诱变结果支持了163和275这两个新突变位点对克拉维酸失活的抗性。这些突变体的分子特征表明，临床分离的大肠杆菌产生的抗抑制剂TEM酶编码基因存在很大差异。
DNA-DNA hybridization and sequencing were performed to determine the molecular basis of resistance to clavulanic acid in 107 inhibitor-resistant TEM (IRT) enzymes produced by Escherichia coli clinical isolates. These beta-lactamases derived from TEM-1 enzyme focused at pI 5.2 (n = 68) or 5.4 (n = 39) and were very poorly inhibited by clavulanic acid compared with TEM-1 enzyme. Results showed that the amino acid sequences of 84 of the 107 enzymes differ from TEM-1 by one or two substitutions previously described: Arg-244-->Ser (IRT-2) in 22 strains, Met-69-->Leu (TEM-33) in 17 strains, Met-69-->Val (TEM-34) in 14 strains, Met-69-->Ile (IRT-3) in 6 strains, Met -69-->Leu associated with Asn-276--> Asp (IRT -4) in 13 strains, and Met-69-->Val associated with Asn-276-->Asp (TEM-36) in 12 strains. A new combination, Met-69-->Ile with Asn-276-->Asp, was found in 20 strains and was called IRT-8, Two IRT enzymes not previously described were characterized, The substitution Met-69-->Val associated with a novel substitution Arg-275-->Leu occurred in one strain, The combination Met-69-->Leu and Asn-276-->Asp was associated with the novel substitution Trp-165-->Arg in two strains, These two novel enzymes were called IRT-9 and IRT-10, respectively. The implication of these novel mutated positions, 163 and 275, in resistance to inactivation by clavulanate was supported by crystallographic data on the TEM-1 enzyme and results of site-directed mutagenesis. Molecular characterization of these mutants showed great diversity among the genes coding for inhibitor-resistant TEM enzymes produced by clinical E. coli isolates.