耐碳青霉烯 鲍曼

中南大学学报（医学版）

J Cent South Univ (Med Sci)2012, 37(5) ; 521长沙地区临床分离碳青霉烯类耐药鲍曼不动杆菌的

分子流行病学特征

梁伟1,2，邹明祥1，邬靖敏1 , 邬国军3，李军1，豆清娅1 ，刘文恩1

(1. 中南大学湘雅医院检验科，长沙 410008；2. 长沙市第三人民医院检验科，长沙 410015；

3. 中南大学基础医学院微生物学系，长沙 410078)

[摘要]目的：了解长沙地区临床分离鲍曼不动杆菌的耐药性，探讨碳青霉烯类抗生素耐药菌株的分子流行病学特征。方法：收集长沙地区10所综合性医院2010年3月至2010年12月间临床分离的非重复鲍曼不动杆菌株205株；采用K-B法检测药物敏感性，改良双纸片协同试验检测金属β-内酰胺酶(金属酶)，改良Hodge试验筛查碳青霉烯酶；PCR扩增OXA-23，OXA-24，OXA-51，OXA-58及IMP-1和VIM-2型碳青霉烯酶基因，并进行测序分析。应用肠杆菌科基因间一致重复序列聚合酶链反应(enterobacterial repetitive intergenic consensus PCR，ERIC-PCR)对菌株进行DNA 分型及同源性分析。结果：在监测的18种药物中，耐药率超过50%的达14种。其中哌拉西林耐药率最高(80.5%)，头孢哌酮/舒巴坦耐药率最低(2.5%)。共筛选出耐碳青霉烯类药物鲍曼不动杆菌115 株，其金属酶表型及基因检测均为阴性；改良Hodge试验阳性71株，其中64株OXA-23基因扩增阳性。115株菌株OXA-51均阳性，未检出OXA-24，OXA-58基因。115株菌株共分为7个ERIC基因型。其中A型19株，B型72株，为主要的流行克隆。结论：长沙地区临床分离鲍曼不动杆菌多重耐药十分严重；产OXA-23 和OXA-51型碳青霉烯酶是鲍曼不动杆菌对碳青霉烯类药物耐药的重要机制，且碳青霉烯类耐药菌株存在克隆流行。

[关键词]鲍曼不动杆菌；碳青霉烯酶；肠杆菌基因间一致重复序列聚合酶链反应；分子流行病学特征

DOI:10.3969/j.issn.1672-7347.2012.05.018 Molecular epidemiological characteristics of clinically isolated carbapenem-resistant Acinetobacter baumannii

in Changsha

LIANG Wei1,2, ZOU Mingxiang1, WU Jingmin1, WU Guojun3, LI Jun1, DOU Qingya1, LIU Wenen1

(1. Department of Clinical Laboratory, Xiangya Hospital, Central South University, Changsha 410008;

2. Department of Clinical Laboratory, Third Hospital of Changsha, Changsha 410015;

3. Department of Microbiology, School of Basic Medicine, Central South University, Changsha 410078, China)

ABSTRACT Objective: To survey antibiotic resistance of clinical isolates of Acinetobacter baumannii in Changsha and to investigate molecular epidemiological characteristics of carbapenem-resistant

Acinetobacter baumannii.

Methods: A total of 205 non-duplicated, clinical isolates of Acinetabacter baumannii from 10 general

hospitals in Changsha were collected from March 2010 to December 2010. The K-B disk diffusion

收稿日期(Date of reception)：2012–01–21

作者简介(Biography)： 梁伟，硕士，主管检验师，主要从事细菌耐药机制研究。

通信作者(Corresponding author)：邹明祥，Email: zoumingxiang@

中南大学学报(医学版), 2012, 37(5) ; 522

鲍曼不动杆菌是引起医院感染的重要病原菌，其分离率位居我国临床常见革兰阴性杆菌第三位，仅次于大肠埃希菌和肺炎克雷伯菌[1]。碳青霉烯类抗生素是治疗鲍曼不动杆菌严重感染的主要药物，但由于该类抗生素在临床上的广泛应用，鲍曼不动杆菌对其耐药率不断增加，现已成为全球严重的公共卫生问题，也是我国目前最重要的“超级细菌”[2–3]。为了解长沙地区临床分离鲍曼不动杆菌的耐药现状，探讨碳青霉烯类抗生素耐药菌株的分子流行病学特征，笔者对长沙地区10所综合性医院临床分离的鲍曼不动杆菌进行了相关研究，现报告如下。

1 材料与方法

1.1材料

1.1.1 临床菌株

收集2010年3月至2010年12月从长沙地区10家综合性医院(中南大学湘雅医院，中南大学湘雅二医院，中南大学湘雅三医院，湖南省第二人民医院，长沙市中心医院，长沙市第一医院，长沙市第三医院，长沙市第四医院，湖南旺旺医院，浏阳市人民医院)各类临床标本中分离的205株非重复鲍曼不动杆菌。所有菌株经法国梅里埃API细菌鉴定系统或Vitek2全自动微生物鉴定系统鉴定。

1.1.2 质控菌株

大肠埃希菌ATCC25922和铜绿假单胞菌ATCC27853购自卫生部临床检验中心。肺炎克雷伯菌ATCC700603购自湖南省临床检验中心。

1.1.3 抗菌药物

用于K–B法药敏试验的抗菌药物共18种：氨苄西林/舒巴坦、环丙沙星、甲氧苄啶/磺胺甲恶唑、米诺环素、多西环素、四环素、庆大霉素、阿米卡星、头孢他啶、头孢吡肟、头孢哌酮/舒巴坦、头孢噻肟、妥布霉素、亚胺培南(imipenem，IMP)、美罗培南(meropenen，MEM)、左旋氧氟沙星、哌拉西林、哌拉西林/他唑巴坦。药敏纸片均为英国Oxiod公司产品。

1.1.4 主要仪器及试剂

主要仪器：基因扩增仪(美国Bio-rad公司)；凝胶成像系统(法国VL公司)；DYY-7型电泳仪(北京市六一仪器厂)。主要试剂：M–H培养基(杭州天和微生物试剂有限公司)；琼脂糖(西班牙Biowest公司)； UNIQ柱式细菌基因提取试剂盒(上海生工生物技术有限公司)；PCR 产物纯化及回收试剂盒(安比奥生物技术公司)；PCR试剂、DNA marker d2000及pGM-T克隆试剂盒(北京天根生化科技有限公司)。

method was applied for the drug-susceptibility test; a modified, double-disk synergy test was used

to detect metallo-β-lactamase (MBL), and a modified Hodge test was used for the screening of

carbapenemase. PCR was used to amplify carbapenemase genes (including OXA-23, OXA-24,

OXA-51, IMP-1, and VIM-2) and the positive products were sequenced. Enterobacterial repetitive

intergenic consensus PCR (ERIC-PCR) was used for DNA typing and test of homology.

Results: Of the 18 antibiotics tested, 14 had a high rate of resistance (> 50% of the isolates tested),

with piperacillin the highest (80.5% of strains), and cefoperazone/sulbactam the lowest (2.5%).

In total, 115 carbapenem-resistant Acinetobacter baumannii strains were confirmed, but their MBL

phenotype and genes were all negative. Seventy-one positive strains were detected by the modified

Hodge test, among which 64 strains were OXA-23-positive. All the 115 strains were positive for

the amplification of the OXA-51 gene, and no strain was found which carried OXA-24 or OXA-58

gene. Seven genomic types were included in the 115 Acinetobacter baumannii. The major prevalence

types were Type B ( 72 strains) and Type A (19 strains).

Conclusion: Multiple drug resistance of clinically isolated Acinetobacter baumannii is a serious

problem in Changsha. Production of OXA-23 and OXA-51 carbapenemases is an important

mechanism of resistance to carbapenem antibiotics, and there is prevalence of the same clones in

these carbapenem-resistant strains.

KEY WORDS Acinetobacter baumannii; carbapenemases; enterbacter repetitive intergenic consensus PCR;

molecular epidemiological characteristics