碳青霉烯类耐药的不动杆菌分子流行病学及其泛耐药的分子机制

Molecular epidemiology and resistant mechanisms of carbapenem-resistant Acinetobacter species

目的调查不动杆菌属对碳青霉烯类的耐药性,研究碳青霉烯类耐药的不动杆菌(CRAB)分子流行病学及耐药机制。方法收集2003—2004年我国10家教学医院187株鲍曼不动杆菌,采用琼脂稀释法确定抗菌药物的最低抑菌浓度(MIC);采用脉冲场凝胶电泳(PFGE)和随机引物多态性DNA(RAPD)分型对128株CRAB进行分子流行病学研究;对多种β内酰胺酶基因进行聚合酶链反应(PCR)及序列分析;对整合子可变基因进行PCR及序列分析。12%十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析CRAB菌株的外膜孔通道蛋白(OMP)。体外筛选亚胺培南的耐药突变子。结果多中心的数据显示,碳青霉烯类耐药率从2003年的4·5%升至2004年的18·2%,多重耐药菌株从2003年的33%升至2004年的48%。北京、广州、福州、杭州、济南的7家医院均发现CRAB耐药克隆的暴发流行。2004年北京协和医院发生泛耐药株的暴发,波及18个病房的74例患者,59·4%的患者(44例)为感染,主要引起肺部感染和菌血症,其中菌血症的病死率40·2%。耐药克隆株均产多种β内酰胺酶:OXA-23型碳青霉烯酶或IMP-8型金属酶、PER-1型酶、高产AmpC酶和TEM-1酶。整合子含有携带氨基糖苷类、利福平、氯霉素的修饰酶及金属酶(blaIMP-8)。不同克隆株OMP差异很大。利血平试验未发现外排机制的高表达。体外成功选择出亚胺培南的耐药突变子,OMP20000的缺失、OMP29000和OMP21000的表达降低是突变子耐药的主要原因。头孢哌酮/舒巴坦、黏菌素、米诺环素可以联合用于CRAB的治疗。结论CRAB克隆株的传播是造成中国多家医院碳青霉烯类耐药率增加的重要原因。不动杆菌通过产生碳青霉烯酶、不同基因盒的整合子、外膜孔蛋白多个通道的缺失,共同介导多重耐药性或泛耐药性。CRAB株引起的感染预后差,必须采取有效的感染控制措施和抗菌药物的干预政策以控制耐药株的传播。

Objective To investigate antimicrobial resistance in Acinetobacter species and molecular epidemiology and resistance mechanism of carbapenem-resistant Acinetobacter species （CRAB）. Methods Non-repetitive 187 carbapenem-resistant isolates of Acinetobacter baumannii were collected from 10 teaching hospitals from 2003 to 2004. Agar dilution method was used to determine the minimal inhibitory concentration （MICs） of the antibiotics. The homology of 128 isolates of CRAB was determined by both pulsed field gel electrophoresis （PFGE） and randomly amplified polymorphism DNA （RAPD）. The betalactamases genes were amplified and sequenced. Class 1 integrons were amplified and sequenced. Outer membrane protein （OMP） of these clones were analyzed by SDS-pelyacrylamide gel electrophoresis （SDS-PAGE）. In vitro selection of sub-MIC concentration of imipenem was performed for mutants. Results Resistance rate of carbapenems among Acinetobacter baumannii increased from 4.5 % in 2003 to 18.2% in 2004 at 10 teaching hospitals in China. The prevalence of multiple resistances to three or more different kinds of antibiotics increased from 33% in 2003 to 48% in 2004. Outbreaks of CRAB clone occurred at 7 hospitals in Beijing, Guangzhou, Fuzhou, Hangzhou and Jinan. In 2004, the outbreak caused by pan-drug resistant clone occurred at Peking Union Medical College Hospital. Seventy-four patients from 18 different wards were infected or colonized by this clone. Almost 59. 4% of cases （44 patients） were infected. The most common nosocomial infection was pneumonia and bacteremia. The mortality of bacteremia was 40. 2%. Resistance clones produced multiple β-1actamases, including OXA-23 carbapenemase or IMP-8 metallo enzyme, PER-1 enzyme high-level AmpC and TEM-1. Integrons harbored the genes hydrolyzing aminoglycosides, rifampin, chloramphenicol and carbapenems （blaIMP-8）. Different OMP patterns were found in different clones. No high expression of efflux pump was detected by reserpine inhibition. Under in vitro selection of imipenem, mulptile resistant mutants were selected. Deletion of OMP 200 00 and reduced expression of OMP 29 000 and OMP 21 000 were the main mechanism of resistance to multiple drugs. Combination of cefoperazone/sulbactam, colistin and minocycline can be used to treat the infection of CRAB. Conclusions The spread of CRAB clone resulted in the increasing trend of carbapenem resistance. The molecular bases of resistance to multiple drugs or pan-drugs in Acinetobacter species included production of carbapenemases, integrons with different resistance gene cassettes and the deletion or reduced expression of OMP. The nosocomial infection caused by CRAB had poor prognosis. Effective infection control measure and antimicrobial intervention policy should be conducted in order to control the outbreak of CRAB.