耐药鲍氏不动杆菌的耐药相关基因检测与聚类分析

Detection of resistance-related genes in drug-resistance Acinetobacterbaumanniiand cluster analysis

目的调查耐药鲍氏不动杆菌菌株间的亲缘关系,对医院感染实时监测,为控制医院感染提供依据。方法收集2011年1-12月医院ICU患者痰液标本中分离的耐药鲍氏不动杆菌共25株,采用聚合酶链反应(PCR)法分析3种与耐药相关的基因(carO、gyrA、parC)和55种水平转移获得与β-内酰胺类、氨基糖苷类、喹诺酮类耐药相关基因以及12种接合性质粒、转座子、插入序列、整合子等可移动遗传元件遗传标记,再对检测结果作聚类分析。结果 25株耐药鲍氏不动杆菌共检出3种与耐药相关的基因(carO、gyrA、parC),4种获得性β-内酰胺类耐药基因(TEM-1、ADC-30/63/64、OXA-23、OXA-66),5种获得性氨基糖苷类耐药基因(aac(3)-Ⅰ、aac(6′)-Ⅰb、ant(3")-Ⅰ、aph(3′)-Ⅰ、armA),2种抗菌制剂外排泵基因(adeB、qacE△1),6种可移动遗传元件的遗传标记(intⅠ1、tnpU、tnp513、IS26、IS903、ISaba1);25株基因carO测得序列均一致,carO基因测得序列与鲍氏不动杆菌敏感株SDF相比存在有义突变;carO基因的氨基酸序列与SDF株相比,一致率为76.0%,并存在3个氨基酸的缺失;gyrA基因有21株存在第83位密码子突变,parC基因有21株存在第80位密码子突变。结论样本聚类分析提示,1～20号株为克隆传播,为泛耐药特征;21～25号株为耐药特征;获得菌株间的亲缘关系对医院感染实时监测和控制医院感染意义重大。

OBJECTIVE To investigate the phylogenetic relationship among agroup of drug-resistant Acinetobacter baumannii（A.Baumannii）strains so as to provide bases for real-time monitoring and control of nosocomial infections.METHODS Totally 25strains of drug-resistant A.baumannii were collected from the sputum specimens that were obtained from the ICU patients from Jan to Dec,2011,then three drug resistance genes（carO,gyrA, parC）,55kinds of resistance genes acquired by horizontal transfer to beta-lactams,aminoglycosides,and quinolones,as well as 12kinds of genetic markers of conjugal plasmids,transposons,insertion sequences,and integrons were detected by means of polymerase-chain-reaction（PCR）assay,and the cluster analysis of the detection result was performed.RESULTS In the 25strains of drug-resistant A.baumannii,3kinds of housekeeping genes（carO, gyrA,parC）,4kinds of acquired beta-lactam-resistance genes（TEM-1,ADC-30/63/64,OXA-23,OXA-66）,5 kinds of acquired aminoglycoside-resistance genes（aac（3）-Ⅰ,aac（6′）-Ⅰb,ant（3＂）-Ⅰ,aph（3′）-Ⅰ,armA）, 2kinds of drug efflux genes（adeB,qacE△1）,and 6kinds of genetic markers of mobile genetic elements（intⅠ1, tnpU,tnp513,IS26,IS903,ISaba1）have been detected positive,moreover,the sequencing analysis showed that 25strains carried the same gene of carOand had missense mutations compared with sensitive strain SDF,and the accordance rate of amino acid sequence of CarObetween the strains and strain SDF was 76.0%,and differences existed due to loss of 3amino acids.In addition,mutations at 83rd codon existed in 21 gyrAgene and mutations at 80rd codon existed in 21 parCgenes.CONCLUSIONThe sample cluster analysis indicates strains 1-20are the same clone and are pandrug-resistant strains and that strains 21-25are drug-resistant strains;it is of great significance in the real-time monitoring and control of nosocomial infections to understand the phylogenetic relationship among the acquired strains.