不动杆菌属多重耐药及泛耐药的分子机制研究

目的 调查B内酰胺酶、整合子、外膜蛋白（Omp）及外排泵在多重耐药和泛耐药不动杆菌属中所起的作用。方法 收集1999至2004年北京、广州和福州对碳青霉烯类耐药的非重复的90株菌。脉冲场凝胶电泳（PFGE）和随机引物多态性DNA（RAPD）分型确定这些耐药株的亲缘关系，选取7个不同克隆深入研究其机制。等电聚焦电泳测定酶的等电点；碱裂解法提取质粒；对各种B内酰胺酶TEM、SHV、PER、OXA-、IMP-、VIM-进行聚合酶链反应（PCR）及序列分析；对整合子基因进行PCR及序列分析。结果 2004年北京协和医院发生泛耐药株P克隆的暴发流行，此克隆株对常用广谱抗菌药物均不敏感。7个不同克隆株均产多种B内酰胺酶：TEM-1酶、高产AmpC酶、SHV型酶、OXA-23型碳青霉烯酶（6株）及IMP-8型金属酶（1株），1个克隆株还产PER-1型酶。这7个克隆株对大多数超广谱6内酰胺类（包括碳青霉烯类）、红霉素、氯霉素、利福平均耐药，2个克隆株对头孢哌酮／舒巴坦、阿米卡星敏感，4个克隆株对左氧氟沙星敏感。所有7株对米诺环素、黏菌素敏感。发现5种不同结构的整合子，携带水解氨基糖苷类、利福平、氯霉素及碳青霉烯类（bla1MR-8）的耐药基因。结论 不动杆菌产生OXA-23型碳青霉烯酶或IMP型金属酶、通过基因重组获得各种携带不同基因盒的整合子，共同介导其多重耐药性或泛耐药性。泛耐药株对个别不常用的抗菌药物如黏菌素、米诺环素仍敏感。

Molecular mechanism of multiple-drug and pan-drug resistance among Acinetobacter species

OBJECTIVE: To investigate the molecular mechanism of multiple-drug and pan-drug resistance among Acinetobacter species. METHODS: Non-repetitive 90 carbapenem-resistant strains of Acinetobacter species were collected in Beijing, Guangzhou, and Fuzhou 1999-2004. The homology of the isolates was determined by both pulsed field gel electrophoresis and randomly amplified polymorphic DNA typing. Seven representative clones were selected from the 90 strains of Acinetobacter isolated from different hospitals to be used for further study. Analytical isoelectric focusing was used to measure the isoelectric point of the beta-lactamase. Plasmid DNA was extracted and purified Genes of different beta-lactamase, including bla(TEM--), bla(SHV-), bla(PER-), blaI(MP-), bla(VIM-), and bla(OXA-) genes, in these clone strains were amplified and sequenced. PCR was used to analyze the integrons. RESULTS: The P clone strain isolated during an outbreak of pan-drug-resistant Acinetobacter species in Peking Union Medical College Hospital 2004 was not susceptible to most common antimicrobial agents tested. The 7 representative clones produced multiple beta-lactamases: TEM-1, high-level AmpC, SHV-type, OXA-23 carbapenemase and IMP-8 and metalloenzyme respectively. One clone produced PER-1 enzyme. These 7 clone strains were resistant to most beta-lactams (including carbapenems), erythromycin, chloramphenicol, and rifampin. Two clone strains were susceptible to cefoperazone/sulbactam and amikacin while 4 clone strains susceptible to levofloxacin. All of the 7 clones were susceptible to minocycline and colistin. Five different integrons were found, harboring the genes mediating the resistance to aminoglycosides, rifampin, chloramphenicol, and carbapenems (bla(IMP-8)). CONCLUSION: The molecular bases of multiple-drug or pan-drug resistance in Acinetobacter species include production of OXA-23 carbapenemase or IMP type metalloenzyme and integrons with different resistance gene cassettes. Pan-drug-resistant Acinetobacter species are susceptible to old antimicrobials agents, such as colistin and minocycline.