摘要目的 了解浙江部分地区碳青霉烯类耐药肺炎克雷伯菌( CRKP)对氨基糖苷类抗生素(AGs)耐药情况,揭示耐药基因携带情况及耐药菌株分子流行病学规律.方法 收集2013年1月至2014年6月浙江省4家医院分离的CRKP菌株104株,包括浙江大学医学院附属邵逸夫医院(简称S医院)56株,浙江大学医学院附属第一医院(简称Z医院)22株,义乌市中医医院(简称Y医院) 13株和杭州市富阳区第一人民医院(简称F医院)13株.采用VITEK 2 Compact法和K-B纸片法检测菌株对常用药物及3种AGs(卡那霉素、庆大霉素和阿米卡星)进行药敏分析.通过PCR及测序技术筛查常见氨基糖苷类耐药基因:16S rRNA甲基化酶基因(rmtA、rmtB和armA)及氨基糖苷修饰酶耐药基因[aac(6′)Ⅰb],并分析耐药性与耐药基因携带状态的关系.采用PFGE对rmtB阳性菌株进行同源性分析,解析各医院菌株流行播散规律.结果 104株CRKP菌株均为多重耐药菌,对头孢菌素类、氟喹诺酮类中的环丙沙星和左旋氧氟沙星及呋喃妥因均具有极高的耐药率,对庆大霉素、卡那霉素和阿米卡星的耐药率分别为:73.1%(76/104)、64.4%(67/104)和56.7%(59/104);氨基糖苷类耐药基因的携带率分别为rmtB 56.7%(59/104)、aac(6′)Ⅰb 17.3%(18/104)、armA 1.9%(2/104),未检到rmtA,未携带筛选基因的菌株有37株.阿米卡星耐药株同时对卡那霉素和庆大霉素耐药,且均为rmtB阳性菌株. PFGE分型结果显示,104株菌分为11个克隆群,每家医院均存在散在分布的非流行克隆.携带rmtB基因菌株有7个主要流行克隆群(Ⅰ～Ⅶ),其中Ⅰ型、Ⅲ型和Ⅴ型流行于S医院,Ⅱ型、Ⅵ型和Ⅶ型流行于Z医院,Y医院的菌株呈克隆散在分布,F医院有Ⅳ型克隆型别(3株).结论 AGs对CRKP菌株仍有一定敏感性,菌株对AGs耐药产生的主要机制是rmtB基因介导的16S rRNA甲基化酶,医院须加强院感防控.

abstractsObjective To analyze the aminoglycoside ( AG ) antibiotics resistance rate of carbapenem-resistant Klebsiella pneumoniae ( CRKP ) and its molecular mechanisms.Methods One hundred and four strains of CRKP isolated from 4 hospitals in Zhejiang Province from January 2013 to June 2014 were collected, including 56 strains from Sir Run Run Shaw Hospital , Zhejiang University School of Medicine ( S hospital ), 22 from the First Affiliated Hospital, Zhejiang University School of Medicine ( Z hospital), 13 from Yiwu TCM Hospitals (Y Hospital) and 13 from Fuyang First People's Hospital (F Hospital).VITEK 2 Compact method and K-B disk method were used to detect the susceptibility of commonly used antibiotics including three kinds of AGs (kanamycin, gentamycin and amikacin ).PCR and sequencing techniques were used to screen the aminoglycoside resistance -related 16S rRNA methylation genes (rmtA, rmtB and armA) and the aminoglycoside modified enzyme resistance gene [aac(6′)Ⅰb].The relationship between drug resistance and carrier status of drug resistance genes was analyzed .Homologous analysis of rmtB-positive strains was performed using PFGE to examine the epidemic spread of strains in each hospital.Results All 104 CRKP strains were multi-drug resistant and had high resistance to cephalosporins, fluoroquinolones ( ciprofloxacin, levofloxacin ) and nitrofurantoin.The resistance rates to gentamicin, kanamycin and amikacin were 73.1%(76/104), 64.4%(67/104) and 56.7%(59/104), respectively.The carrying rates of aminoglycoside-resistance genes were: rmtB 56.7%( 59/104 ), aac (6′)Ⅰb 17.3%(18/104), armA 1.9%(2/104); while no rmtA was detected.Thirty-seven strains did not carry the screened genes.Amikacin-resistant strains were resistant to both kanamycin and gentamicin, and both were rmtB-positive strains.The PFGE classification results showed that 104 strains were divided into 11 clonal populations, and there were scattered non-population clones in each hospital. There were seven major clonal populations (Ⅰ-Ⅶ) carrying rmtB genes, of which typeⅠ, typeⅢand typeⅤwere prevalent in S hospital ; typeⅡ, typeⅥand TypeⅦwere popular in Z hospital ; the distribution of strains in Y hospital was scattered ; F hospital had one independent clone type Ⅳ(3 strains).Conclusion AGs still have certain sensitivity to CRKP strains.The main mechanism of strain resistance to AGs is the rmtB gene-mediated 16S rRNA methylase.