棒杆菌属细菌药敏试验方法学评价及对喹诺酮类药物主要耐药机制的研究  

Evaluation of susceptibility testing for Corynebacteriumand investigation of resistant mechanisms to quinolones

作　　者：杨雪静[1] 曹俊敏[1] 张伟珍[1] 王原[1] 

机构地区：[1]浙江中医药大学附属第一医院检验科,浙江杭州310006

出　　处：《中华医院感染学杂志》2013年第6期1235-1238,共4页Chinese Journal of Nosocomiology

基　　金：浙江省教育厅资金资助(2009E0901)

摘　　要：目的筛选适合临床常规使用的检测棒杆菌属药敏试验的方法,分析棒杆菌属的耐药状态,为临床合理使用相关抗菌药物提供依据;探讨gyrA、parC基因的改变与棒杆菌属耐喹诺酮类抗菌药物的关系。方法采用K-B纸片扩散法、琼脂稀释法、E-test、VITEK-AMS、微量稀释法等5种方法测试纹带棒杆菌及假结核棒杆菌对16种抗菌药物的药物敏感性;PCR扩增检测gyrA和parC基因喹诺酮类耐药决定区相关片段并测序,在GenBank中进行Blast及BlastX分析并观察氨基酸突变点,用NcoI酶进行PCR-RFLP。结果 5种方法在检测万古霉素药物敏感性无差异,K-B纸片扩散法有极重大误差及重大误差,经Bowker检验,差异有统计学意义(P<0.05);细菌在AST-GP67卡片中未充分生长;37株纹带棒杆菌对环丙沙星和左氧氟沙星的耐药率均为94.6%,30株菌发生双突变(87位Ser突变为phe,91位Asp突变为Ala),5株菌单点突变(87位Ser突变为phe);PCR-RFLP经NcoI酶切后显示,敏感株和耐药株均产生3个条带。结论棒杆菌属对多种抗菌药物的耐药率较高,对万古霉素全部敏感;VITEK-2Compact仪器法、K-B纸片扩散法不适用于棒杆菌属药敏试验,E-test、琼脂稀释法可采纳,但红霉素除外;纹带棒杆菌对喹诺酮类抗菌药物的耐药机制主要是gyrA基因突变引起,尤以双突变占优势,纹带棒杆菌不携带parC基因,不编码拓扑异构酶Ⅳ。OBJECTIVE To screen the methods of susceptibility testing of Corynebacterium and analyze the drug resistance status so as to furnish basis for the reasonable clinical medication; to investigate the relationship between gyrA, parC genes and resistance of Corynebacterium to quinolones. METHODS The K-B disk diffusion method, agar dilution,E-test, VITEK-AMS and mierodilution method were used to detect the antibiotic susceptibility of Corynebacteriurn. The quinolone resistance determining regions（QRDRs） of the gyrA and parC gene of C. striaturn were amplified. The sequences and mutations of gyrA gene were analyzed in GenBank by Blast and BlastX. The PCR product of the gyrA gene was subjected to digestion with NcoI restriction enzyme. RESULTS The methods described above were no difference in vaneomyein. Very major and major errors were encountered when comparing disk diffusion with the others. The growth of Corynebacterium was suppressed in AST-GP67. Resistant rates of C. striatum to eiprofloxacin and levofloxacin was 94.6 ~/I0. Thirty strains presented a double mutation at positions 87 and 91 ,with a change from Set to Phe in the amino acid codon 87 and from Asp to Ala at position 91. Five strains presented a mutation at positions 87. Three bands were obtained after PCR-RELP. CONCLUSION The resistance rates of C. striatum to antibiotics are relatively high. These strains are sensitive to vancomycin. VITEK-2 compact and disk diffusion are not suitable for the detection of the susceptibility testings of Corynebacterium. The agar dilution and E-test can be adopted except for erythromyein. The mutation of gene is the main cause leading to the resistant mechanisms of C. striatum to quinolones, especially the double mutation as dominant. C. striatum lacks the parC gene, without encoding topoismerase IV.

关 键 词：棒杆菌属 药敏试验 喹诺酮类药物 耐药机制 

分 类 号：R378[医药卫生—病原生物学]