摘要
目的探讨临床分离大肠埃希菌RJ904对阿莫西林-克拉维酸、哌拉西林-他唑巴坦等含β内酰胺酶抑制剂抗菌药物和头孢哌酮耐药,而对头孢西丁、头孢噻肟敏感的机制。方法通过三相水解试验了解大肠埃希菌RJ904所产酶的水解特性;以大肠埃希菌J53Azr为受体菌,临床菌株RJ904为供体菌,通过接合试验了解该耐药特性是否由质粒介导;通过"鸟枪法"随机克隆试验将耐药质粒经BamHI和HindⅢ双酶切后的片段插入到克隆载体pACYC184中表达并测序,明确介导该耐药特性的基因;通过重叠延伸法定点突变及亚克隆了解启动子对耐药基因表达的影响。结果菌株RJ904中的酶可水解哌拉西林-他唑巴坦、头孢哌酮,但不水解头孢西丁、头孢噻肟,而且其耐药性可通过质粒接合转移。耐药质粒经BamHI和HindⅢ酶切后重组到载体pACYC184中的3.9 kb双酶切片段,含有大小861 bp的blaTEM-1B基因,其上游的启动子为Pa/Pb。该重组载体表达后对头孢哌酮、哌拉西林-他唑巴坦等含酶抑制剂抗菌药物耐药,而对头孢西丁、头孢噻肟敏感,重组载体中blaTEM-1B基因启动子经32位的T→C定点突变后,由强启动子Pa/Pb变为弱启动子P3,突变后的重组质粒转化入大肠埃希菌DH5α后所表达的耐药性与突变前相比MIC值均有较大下降。结论本试验首次证实在大肠埃希菌中Pa/Pb启动子调控TEM-1β内酰胺酶的高表达可使细菌对阿莫西林-克拉维酸、哌拉西林-他唑巴坦等含酶抑制剂抗菌药物和头孢哌酮耐药,而且这种耐药基因位于转座子和质粒上,可通过接合转移扩散。
Objective To explore the mechanism of resistance to piperacillin-tazobactam and cefoperazone but susceptible to cefotaxime in a cinical isolate of Escherichia coli. Methods The enzymes produced by E. coli RJ904 was characterized by three-dimensional testing. Conjugation experiments were carried out with E. coli J53Az^r as recipient to examine if the resistance gene was plasmid-mediated. The BamⅢ and HindⅢ digested fragments of the transferable plasmid were cloned into the vector pACYC184. The nucleotide sequences of the cloned fragment were analyzed and extended sequence analysis was performed to gain insight into the structure of the resistant gene. The site-directed mutation of the promoter Pa/Pb to P3 was performed by overlap-extension PCR to evaluate the expression of the TEM-1β-laetamase gene. Results The enzyme produced by E. coli RJ904 could hydrolyze piperacillin-tazobactam and cefoperazone but not cefotaxime or cefoxitin. This resistant trait was plasmid medi ated. The BamHI and HindⅢ digested fragment （3.9 kb） cloned into the vector pACYC184 was sequenced and showed a 861 bp blaTEM-1B gene and the upstream promoter Pa/Pb in it. The expression of the above fragment was resistant to piperacillin-tazobactam and cefoperazone. The strong promoter Pa/Pb was mutated to a weaker P3 by T→C point mutation at position 32. The expression of the fragment after such a mutation was susceptible to piperacillin-tazobactam and cefoperazone. Conclusions The hyperproduction of TEM-1β laetamases mediated by promoter Pa/Pb is responsible for the resistance to piperacillin-tazobactam and cefoperazone in E. coli. The resistant gene is located on transoposon and transferable plasmid.
出处
《中国感染与化疗杂志》
CAS
北大核心
2013年第3期167-172,共6页
Chinese Journal of Infection and Chemotherapy
基金
上海市科学技术委员会上海自然科学基金自助项目(11ZR1422200)
