耐药结节细胞分化超家族外排泵介导碳青霉烯类耐药鲍曼不动杆菌对替加环素耐药机制的研究  

Effect of resistance nodulation division efflux pumps on reduced susceptibility to tigecycline in carbapenem-resistant Acinetobacter baumanii

作　　者：沈晓强[1] 陈琼[2] 周华[3] 蒋琰[2] 俞云松[2] 

机构地区：[1]杭州市红十字会医院呼吸,310003 [2]浙江大学医学院附属邵逸夫医院感染科 [3]浙江大学医学院附属第一医院呼吸科

出　　处：《中华临床感染病杂志》2014年第5期387-392,共6页Chinese Journal of Clinical Infectious Diseases

基　　金：国家自然科学基金（81000757）;杭州市科技发展计划项目（20110833B27）

摘　　要：目的 研究碳青霉烯类耐药鲍曼不动杆菌中耐药结节细胞分化超家族（RND）外排泵介导菌株对替加环素敏感性降低的机制.方法 连续收集2010年7个省市16家医院鲍曼不动杆菌631株.采用PCR检测oxa-51和oxa-23基因,并对菌株进行多位点序列分型（MLST）.用纸片药敏法检测β-内酰胺类、氨基糖苷类、大环内酯类、四环素类、碳青霉烯类抗生素、替加环素和多黏菌素的抑菌圈直径.对替加环素抑菌圈直径≤12 mm的菌株采用E-test法检测替加环素最小抑菌浓度（MIC）.使用外排泵抑制剂1-（1-萘甲基）哌嗪（NMP）检测操纵子基因中adeB、adeG和adeJ的表达,确定外排泵抑制剂抑制表型的分布.选取对替加环素和碳青霉烯类均耐药,且具有外排泵抑制剂抑制表型的菌株作为实验组;对替加环素敏感而对碳青霉烯类耐药的菌株作为对照组,并以对替加环素敏感的ATCC1 9606菌株作为标准菌株.采用实时荧光定量PCR （qPCR）检测实验组和对照组外排泵adeABC、adeFGH和adelJK的转录水平并进行比较.采用PCR法对adeR、adeS和adeL进行全基因长度测序,查找突变位点或插入序列.结果 631株鲍曼不动杆菌中,共筛选出32株对替加环素敏感性降低的碳青霉烯类耐药鲍曼不动杆菌,其中8株具有外排泵抑制剂抑制表型.同时选取对替加环素敏感而对碳青霉烯类耐药的4株作为对照组.实验组A518、Z1219和A527菌株adeABC表达明显增高,分别是标准株ATCC19606的13,5和7倍;adeFGH和adeIJK表达量在部分菌株中稍有上调.对照组A207和A1731菌株在转录水平未检测到adeABC,其他菌株adeABC、adeFGH和adeIJK表达没有上调.在adeR和adeS分别发现多态性位点E220K和A130D.在adeABC高表达菌株中发现了adeS的ISAbal插入突变.adeL中未发现突变位点.结论 adeABC高表达在碳青霉烯类耐药鲍曼不动杆菌替加环素耐药机制中发挥重要作用,主要是调控基因adeS中存在ISAbal插Objective To investigate the effect of resistance nodulation division （RND） efflux pumps on reduced susceptibility to tigecycline in carbapenem-resistant Acinetobacter baumanii.Methods Totally 631 isolates of Acinetobacter baumanii were collected from 16 hospitals in 7 provinces in 2010.Genes oxa-51 and oxa-23 were detected by PCR method,and the ST profiles were determined by multilocus sequence typing （MLST）.The disk susceptibility assay was used to determine the inhibition zone diameters of β-lactams,aminoglycosides,macrolides,tetracyclines,carbapenems,tigecycline and polymyxin.The minimum inhibitory concentration （MIC） of tigecycline was determined by E-test in strains with inhibition zone diameters ≤ 12 mm on tigecycline.The expression of operon genes adeB,adeG and adeJ was determined with efflux pump inhibitor NMP （N-methyl-2-pyrrolidone） for detection of efflux pump inhibitor phenotype.The isolates of Acinetobacter baumanii which were resistant both to tigecycline and carbapenems and with the inhibited phenotype of efflux pump inhibitor were collected as the experiment group,the isolates which were susceptible to tigecycline but resistant to carbapenems were collected as the control group,and ATCC 19606 was used as the reference strain.The expressions of adeABC,adeFGH and adeIJK were quantified by q-PCR at the transcriptional level.Genes adeR,adeS and adeL were amplified and sequenced using PCR method to find polymorphic locus and insertion sequences.Results There were 32 isolates of Acinetobacter baumanii with reduced susceptibility to tigecycline and carbapenem-resistant.Eight isolates were with the inhibited phenotype by efflux pump inhibitor.And 4 strains which were susceptible to tigecycline but resistant to carbapenems were selected as the control.The expressions of adeABC in A518,Z1219 and A527 of experiment group were 13-fold,5-fold and 7-fold higher than reference strain ATCC19606,respectively.The expressions of adeFGH and adeIJK were up-regulated slightly in some isolates.Transc

关 键 词：鲍氏不动杆菌 抗药性 多药 耐药结节细胞分化超家族 替加环素 

分 类 号：R446.5[医药卫生—诊断学]