摘要
目的通过运用变性高效液相色谱(DHPLC),研究鲍曼不动杆菌耐药表型与其染色体上gyrA和parC基因突变的关系。方法采用微量肉汤稀释法测定左氧氟沙星和加替沙星对90株临床分离鲍曼不动杆菌的最低抑菌浓度(MIC);采用基因外序列进行同源性分析,PCR扩增鲍曼不动杆菌的gyrA和parC基因,对PCR产物进行DHPLC检测及DNA测序。结果 90株鲍曼不动杆菌中,对左氧氟沙星和加替沙星耐药率分别为63.3%和60.0%;同源性分析可分成9种基因型,90株扩增出了343bp的gyrA基因和327bp的parC基因产物。9种基因型中,3种表型耐药的基因型PCR产物经DHPLC检测均出现异常双峰或者多峰。测序证实gyrA基因存在83位Ser→Leu突变,parC基因在80位同样出现Ser→Leu突变,同时parC基因还存在多个位点的同义突变;敏感株运用DHPLC检测只出现单峰,测序未发现突变。结论运用DHPLC检测可以快速发现细菌耐药基因的突变,鲍曼不动杆菌对喹诺酮类药物的耐药与gyrA和parC基因的高频突变相关。
Objective To study the relations of resistance phenotype of Acinetobacter baumannii and gene mutations of gyrA and parC on the chromosomes by denaturing high performance liquid chromatography （DHPLC）. Methods Micro broth dilution was used to determine the minimum inhibitory coneentrations（MIC） of Levofloxacin and Moxifloxacin of 90 Acinetobacter baumannii isolated from the clinical specimens; Gene homology was analyzed by repetitive extragenic palindrome. Polymerase chain reaction （PCR） was used to amplify the gyrA and parC. The PCR products were analysed by DHPLC and sequencing. Results The resistance of Levofloxaein and Moxifloxacin was 63.3 % and 60.0%in the 90 Acinetobacter baumannii. There were nine genotypes in the clinical isolates. Ninety had the expect sizes of 343bp of gyrA and 327bp of parC. Three out of nine genotypes,which were drug resistant,were all found abnormal elution peaks （two or three peaks） by DHPLC. The result of DNA sequencing demonstrated that mutations in gyrA gene that resulted in amino acid substitutions of Ser83→eu and in parC that resulted in Ser80→Leu. And there were some synonymous mutations in parC gene. All the drug susceptible ones showed single peak,and the results of sequencing had no mutation. Conclusion Detecting gene mutations was rapid by DHPLC. And Resistance among quinolone resistant Acinetobacter baumannii strains is related with high frequency mutations in the gyrA and parC genes.
出处
《国际检验医学杂志》
CAS
2011年第4期448-450,共3页
International Journal of Laboratory Medicine
