耐药鲍氏不动杆菌blaOXA基因与插入序列连锁检测及菌株亲缘性研究

Linkage detection of blaOXA and insertion sequence and investigation of relationship among strains in drug-resistant Acinetobacter baumannii

目的探讨耐药鲍氏不动杆菌的抗菌药物耐药基因及可移动遗传元件遗传标记的携带、blaOXA基因与插入序列连锁与菌株间的亲缘性。方法收集2012年1月-2013年8月ICU患者痰液标本中分离到的25株耐药鲍氏不动杆菌,经看家基因测序作BLAST比对确认菌种,采用聚合酶链反应(PCR)及序列分析的方法分析A^D类共34种β-内酰胺酶基因、15种氨基糖苷类耐药基因、2种喹诺酮类耐药基因、6种可移动遗传元件遗传标记,再做ISaba1与blaOXA-2、blaOXA-23、blaOXA-66连锁检测,扩增有阳性条带者再进行DNA测序验证,测序结果用Chromas作BLAST比对,最后对57种耐药基因测得结果作样本聚类分析。结果 25株耐药鲍氏不动杆菌共检出7种β-内酰胺类耐药基因、4种氨基糖苷类耐药基因、2种喹诺酮类耐药基因、4种可移动遗传元件遗传标记,且阳性率非常高;插入序列与blaOXA基因的连锁检测中,检测到blaOXA-23与ISaba1连锁,但blaOXA-2、blaOXA-66与ISaba1未连锁;样本聚类分析发现,1~18号株为同一克隆,20~25号株亦为同一克隆,19号株为孤立株。结论多药耐药鲍氏不动杆菌中携带的耐药基因导致本组菌对β-内酰胺酶类、氨基糖苷类、喹诺酮类耐药,且耐药基因和耐药表型相对应,样本聚类分析提示1~18号株和20~25号株可能为医院感染菌株。

OBJECTIVE To investigate the distribution of resistant genes and genetic markers of mobile genetic ele‐ments ,linkage detection of blaOX A and insertion sequence ,and relationship among strains in drug‐resistant A cin‐etobacter baumannii .METHODS Totally 25 strains of drug‐resistant A .baumannii isolated from the sputum speci‐mens in the hospital from Jan .2012 to Aug .2013 were collected ,and BLAST algorithm was performed to identify A .baumannii through gene sequencing by house‐keeping genes .Then ,34 kinds of β‐lactamase genes （class A to D） ,15 kinds of resistant genes to aminoglycosides ,2 kinds of resistant genes to quinolones ,6 kinds of genetic markers of mobile genetic elements were detected by PCR . In addition , the linkage detection of ISaba1 to blaOXA‐2 ,blaOXA‐23 ,blaOXA‐66 was detected ,DNA sequencing was performed to confirm positive bands , BLAST algorithm was performed by Chromas .Finally ,the sample cluster analysis was performed on the detection results of the 57 resistant genes .RESULTS Totally 7 kinds of beta‐lactamase genes ,4 kinds of resistant genes to aminoglycosides ,2 kinds of resistant genes to quinolones ,4 kinds of genetic markers of mobile genetic elements were positive ,and positive rates were high .Linkage detection of blaOXA‐23 and ISaba1 was positive ,but linkage detection of blaOXA‐2 ,blaOXA‐66 and ISaba1 was negative .The sample cluster analysis suggested that strain No .1-18 were the same clone ,strain No .20 -25 were the other clone ,Strain No .19 was the isolated clone . CONCLUSION Resistant genes in this group of A . baumannii resulted in resistance to beta‐lactams ,aminoglycosides ,quinolones ,and genotypes and phenotypes were identical .The sample cluster analysis suggested that strain No .1-18 and strain No .20-25 might be strains of nosocomial infection .