鲍曼不动杆菌耐药性快速检测方法的建立与评估

Establish and evaluation of rapid detecting antibiotic resistance in Acinetobacter baumanni

目的评估对实时荧光定量聚合酶链反应(PCR)快速检测鲍曼不动杆菌耐药性方法的效果。方法设计鲍曼不动杆菌(ATCC 19606)外膜蛋白A特异性探针,采用实时荧光定量PCR准确测定63株鲍曼不动杆菌经一定浓度庆大霉素、米诺环素、美罗培南、左氧氟沙星和头孢哌酮/舒巴坦孵育后的生长情况,比较抗菌药物孵育0和6h后鲍曼不动杆菌基因组DNA量,判断鲍曼不动杆菌耐药性,并与微量肉汤稀释法结果进行对比。结果建立的实时荧光定量PCR检测鲍曼不动杆菌耐药性方法与微量肉汤稀释法一致性高,Kappa值为0.879,P<0.05。以微量肉汤稀释法为参考方法,实时荧光定量PCR的符合率达93.97%,灵敏度为92.53%,特异性为95.74%,阳性预测值为96.41%,阴性预测值为91.22%,Youden指数为0.88。结论实时荧光定量PCR与微量肉汤稀释法结果有高度一致性,且前者灵敏度高、特异性强、检测速度快,可用于快速检测鲍曼不动杆菌的耐药性。

Objective To assess the ability of rapid detection of antibiotic resistance in Acinetobacter baumannii by real-time fluorescent quantitative PCR.Methods A real-time fluorescent quantitative PCR assay targeting the conserved region of Acinetobacter baumannii ATCC 19606 outer membrane protein（ompA）gene was used to measure the growth of 63 Acinetobacter baumannii isolates,which were incubated in a certain concentration of gentamicin,minocycline,meropenem,levofloxacin or cefoperazone/sulbactam.The number of genomic DNA copies of the Acinetobacter baumannii incubated in different antibiotics for 6hwere compared with those incubated for 0h.Fold changes were used to determine the resistance in Acinetobacter baumannii,and all the results were compared with those obtained using broth microdilution.Results The real-time fluorescent quantitative PCR assay showed high consistancy with broth microdilution method,in which Kappa value was 0.879,P〈0.05.Compared with the broth microdilution,the global values for consistency was 93.97%,sensitivity was 92.53%,specificity was 95.74%,positive predictive value was96.41%,negative predictive value was 91.22%,and Youden index was 0.88.Conclusion The results obtained by the real-time fluorescent quantitative PCR were highly concordant with those of broth microdilution.The real-time fluorescent quantitative PCR for antibiotic resistance detection in Acinetobacter baumannii was characterized by its sensitivity,specificity and rapidity.