质粒介导喹诺酮类qnr基因在肠杆菌科细菌中的流行及耐药特性研究

SPrevalence of plasmid-mediated quinolone resistance gene qnr in Enterobacteriaceae species

目的 了解质粒介导喹诺酮耐药qnr基因在临床分离的三种常见肠杆菌科细菌中的分布,研究qnr阳性株的质粒特性和流行播散情况.方法 VITEK-60全自动微生物分析仪检测大肠埃希菌、肺炎克雷伯菌和阴沟肠杆菌对临床常用抗菌药物的耐药性,琼脂稀释法检测环丙沙星、诺氟沙星和左氧氟沙星的最低抑菌浓度（MIC）;PCR扩增质粒介导的喹诺酮类耐药基因 （qnrA、qnrB、qnrC、qnrD、qnrS）,阳性产物进行DNA测序比对以确定基因型;.对qnr基因阳性菌株进行接合转移试验,探讨qnr基因的可转移性;脉冲场凝胶电泳技术（PFGE）分析同源性及流行播散情况.结果 301株大肠埃希菌中共检出qnr阳性菌株14株,其中2株同时携带两种基因,qnrA、qnrB、qnrS基因分别检出5、2、9株;145株肺炎克雷伯菌中共检出qnr阳性菌株17株,其中1株同时携带两种基因,qnrA、qnrB、qnrS基因分别检出1、11、6株;173株阴沟肠杆菌中共检出qnr阳性菌株57株,其中3株同时携带两种基因,qnrA、qnrB、qnrS基因分别检出14、36、10株;所有菌株中均未检出qnrC和qnrD基因;接合转移试验结果显示,14株qnr基因阳性的大肠埃希菌、17株qnr阳性肺炎克雷伯菌和57株qnr阳性阴沟肠杆菌中分别有4、3和26株体外接合成功,qnrB阳性阴沟肠杆菌中少数菌株存在同源性,大多呈散发流行趋势.结论 临床肠杆科细菌中存在qnr阳性菌株的流行,总体以qnrB为主;qnr基因在喹诺酮类耐药和敏感菌株中均存在,喹诺酮类药物敏感菌株中qnr基因的检出值得关注;qnr基因可通过其所在质粒进行水平转移;携带qnrB基因的阴沟肠杆菌大多无同源性,呈散发流行趋势.

Objective To investigate the prevalence of plasmid-mediate quinolone resistance gene （PMQR） qnr in Enter- obacteriaceae species. Methods The resistance aganist commonly used antibiotics of Escherichia coil, Enterobacter cloacae and Klebsiella Pneumoniae was detected by VITEK-60 AMS; the minimal inhibitory concentration （MIC） of ciprofloxacin, nor- floxacin and levofloxacin was assayed by agar dilution method; the PMQR genes qnrA, qnrB, qnrC, qnrD and qnrS were detected by polymerase chain reaction （PCR） and verified by DNA sequencing analysis. The conjugation test was performed to investigate the transferability of qnr gene and the pulsed-field gel electrophoresis （PFGE） was used to determine the homogeneity and epiclemiological relatedness. Results The qnr genes were detected in 14 out of 301 strains of Escherichia coil; qnrA, qnrB and qnrS genes were detected in 5, 2 and 9 strains respectively and 2 stains concurrently carried two qnr genes. The qnr genes were detected in 17 out of 145 strains of Klebsiella Pneumoniae; qnrA, qnrB and qnrS genes were present in 1, 11 and 6 strains respectively and one stain carried two qnr genes. The qnr genes were detected in 57 out of 173 Enterobacter cloa- cae; qnrA, qnrB, qnrS genes were present in 14, 36 and 10 strains respectively and 3 stains carried two qnr genes. In this study qnrC and qnrD genes were not detected in all Enterobacteriaceae strains. In qnr positive strains, there were 4 stains of Es- cherichia coil, 3 Klebsiella Pneumoniae and 26 Enterobacter cloacae were conjugated by plasmids. In 36 qnrB positive strains of Enterobacter cloacae, only 6 couples were verified as epidemiological relatedness. Conclusion There is an emergence of qnr-borne Enterobacteriaceae species clinically and qnrB was most prevalent in qnr. The qnr genes were detected in both quinolone-resistant and susceptible isolates; the transmission of qnr genes is mainly mediated by transferable plasmids.