CTX-M-38型超广谱β-内酰胺酶的表达、定位及其抗药活性分析

The Analysis of Expression and Antibiotic Susceptibilities of CTX-M-38 Type Extended-Spectrum-Lactamase

目的:体外表达CTX-M-38型超广谱β-内酰胺酶(ESBL),并明确表达产物的分布及其抗药活性。方法:收集2006—2007年分离的产ESBL大肠埃希菌46株,采用PCR技术克隆目的基因,基因重组技术构建pET28a-CTX-M-38质粒,用BL21大肠杆菌作为宿主菌进行表达,测定培养基上清及菌体裂解液酶活性检测所表达ESBL的分布;采用液体稀释法检测所表达ESBL抗药活性。结果:PCR扩增条带约在900bp位置处;测序结果与预测CTX-M-38相一致;菌体裂解液抗药活性较强;转化子对青霉素类、头孢一代、二代及多数三代药物均耐药;对碳青酶烯类药物稳定敏感;对头孢他啶及氨曲南体外敏感;对酶抑制剂除哌拉西林/他唑巴坦敏感外,其余表现为耐药;对庆大霉素、美满霉素、环丙沙星及左氧氟沙星亦具有耐药性。结论:CTX-M-38型ESBL表达成功,表达产物主要分布于表达菌体内,具有广泛抗药活性。

Objective： To express CTX-M-38 type extended-spectrum-lactamase, and detect its distribution and antibi- otic susceptibilities. Methods： Total of 46 strains producing ESBL E.coli was collected from the first affiliated hospital of Zheng zhou University. The CTX-M-38 ESBL gene was selected by PCR using gene recombination technique to construct pET28a-CTX-M-38. The expression of CTX-M-38 in BL21 E.coli and its antibiotic susceptibilities were carried out by liquid dilution test. Testing the enzyme activities of culture supernatant and bacteria sonicate to reflect its distribution. Results： The size of amplified gene product was about 900 bp. The DNA sequence was matched with the information of gene bank. The en- zyme activities from bacteria sonicate were stronger than the culture supernatant .The transformant was resistance to penicillins, the first, second and third generations of cephalosporins. It was sensitive to imipenem. The transformant was also sensitive to ceftazidime and aztreonam in vitro, and resistance to antibiotics including beta -lactamase inhibitors except piperacillin/ tazobactam. The transformant was also resistance to gentamicin,minocycline, ciprofloxacin and levofloxacin. Conclusion： The CTX-M-38 type ESBL is successfully expressed at designed experimental condition in this study. The product mainly lies in- side the bacteria. The transformant shows wide resistance to antibiotics.