Sirot D, Recule C, Chaibi E B, Bret L, Croize J, Chanal-Claris C, Labia R, Sirot J
Laboratoire de Bacteriologie-Virologie, Faculté de Médecine, Clermont-Ferrand, France.
Antimicrob Agents Chemother. 1997 Jun;41(6):1322-5. doi: 10.1128/AAC.41.6.1322.
Escherichia coli GR102 was isolated from feces of a leukemic patient. It expressed different levels of resistance to amoxicillin or ticarcillin plus clavulanate and to the various cephalosporins tested. The double-disk synergy test was weakly positive. Production of a beta-lactamase with a pI of 5.6 was transferred to E. coli HB101 by conjugation. The nucleotide sequence was determined by direct sequencing of the amplification products obtained by PCR performed with TEM gene primers. This enzyme differed from TEM-1 (blaT-1B gene) by four amino acid substitutions: Met-->Leu-69, Glu-->Lys-104, Gly-->Ser-238 and Asn-->Asp-276. The amino acid susbstitutions Leu-69 and Asp-276 are known to be responsible for inhibitor resistance of the IRT-4 mutant, as are Lys-104 and Ser-238 substitutions for hydrolytic activity of the extended-spectrum beta-lactamases TEM-15, TEM-4, and TEM-3. These combined mutations led to a mutant enzyme which conferred a level of resistance to coamoxiclav (MIC, 64 microg/ml) much lower than that conferred by IRT-4 (MIC, 2,048 microg/ml) but higher than that conferred by TEM-15 or TEM-1 (MIC, 16 microg/ml). In addition, the MIC of ceftazidime for E. coli transconjugant GR202 (1 microg/ml) was lower than that for E. coli TEM-15 (16 microg/ml) and higher than that for E. coli IRT-4 or TEM-1 (0.06 microg/ml). The MICs observed for this TEM-type enzyme were related to the kinetic constants Km and k(cat) and the 50% inhibitory concentration, which were intermediate between those observed for IRT-4 and TEM-15. In conclusion, this new type of complex mutant derived from TEM-1 (CMT-1) is able to confer resistance at a very low level to inhibitors and at a low level to extended-spectrum cephalosporins. CMT-1 received the designation TEM-50.
大肠杆菌GR102是从一名白血病患者的粪便中分离出来的。它对阿莫西林或替卡西林加克拉维酸以及所测试的各种头孢菌素表现出不同程度的耐药性。双纸片协同试验呈弱阳性。通过接合作用，将一种pI为5.6的β-内酰胺酶的产生转移到了大肠杆菌HB101中。通过使用TEM基因引物进行PCR获得的扩增产物直接测序来确定核苷酸序列。这种酶与TEM-1（blaT-1B基因）有四个氨基酸替换不同：第69位的甲硫氨酸（Met）替换为亮氨酸（Leu）、第104位的谷氨酸（Glu）替换为赖氨酸（Lys）、第238位的甘氨酸（Gly）替换为丝氨酸（Ser）以及第276位的天冬酰胺（Asn）替换为天冬氨酸（Asp）。已知第69位的亮氨酸和第276位的天冬氨酸替换是IRT-4突变体对抑制剂耐药的原因，第104位的赖氨酸和第238位的丝氨酸替换是超广谱β-内酰胺酶TEM-15、TEM-4和TEM-3水解活性的原因。这些组合突变产生了一种突变酶，它赋予对阿莫西林克拉维酸的耐药水平（MIC，64μg/ml）远低于IRT-4（MIC，2048μg/ml）但高于TEM-15或TEM-1（MIC，16μg/ml）。此外，头孢他啶对大肠杆菌接合子GR202的MIC（1μg/ml）低于对大肠杆菌TEM-15的MIC（16μg/ml）且高于对大肠杆菌IRT-4或TEM-1的MIC（0.06μg/ml）。观察到的这种TEM型酶的MIC与动力学常数Km和k(cat)以及50%抑制浓度相关，这些值介于IRT-4和TEM-15所观察到的值之间。总之，这种源自TEM-1的新型复合突变体（CMT-1）能够对抑制剂产生非常低水平的耐药性，并对超广谱头孢菌素产生低水平的耐药性。CMT-1被命名为TEM-50。
