耐药结节细胞分化超家族介导鲍曼不动杆菌替加环素耐药机制研究

Mechanism of resistance nodulation division efflux pumps mediated tigecycline-insensitive multidrugresistant Acinetobacter baumannii

目的分析耐药结节细胞分化超家族(RND)外排泵介导多重耐药鲍曼不动杆菌(MDR-AB)对替加环素敏感性降低的机制。方法收集20152018年哈尔滨医科大学附属第四医院替加环素不敏感的鲍曼不动杆菌21株及MDR-AB替加环素敏感株39株。以微量肉汤稀释法为标准方法,以替加环素不敏感菌株[最小抑菌浓度(MIC)≥2μg/mL]为实验组,替加环素敏感菌株(MIC≤1μg/mL)为对照组。采用聚合酶链反应(PCR)检测鲍曼不动杆菌替加环素敏感性降低相关的RND基因adeB、adeG和adeJ,及其上游调控基因adeS、baeR和baeS,并对adeS扩增产物进行测序,查找插入序列。采用实时荧光定量聚合酶链反应(RT-qPCR)检测实验组和对照组外排泵adeABC、adeFGH、adeIJK、adeRS和baeRS的转录水平并进行比较。结果微量肉汤稀释法确证有12株MDR-AB为替加环素不敏感株,与替加环素敏感性降低相关的RND基因检出率为100%,且在2株菌株中发现了adeS的ISAbaⅠ插入序列。实验组有3株菌株adeABC表达较对照组明显升高,分别为标准菌株鲍曼不动杆菌(ATCC 19606)的3.3、3.5和2.7倍;有1株菌株adeFGH表达量升高明显;adeIJK、adeRS和baeRS表达量在部分菌株中稍有上调。结论RND外排泵介导的MDR-AB对替加环素敏感性降低与adeABC和adeFGH高表达密切相关,adeABC高表达可能与其上游调控基因adeS中存在ISAbal插入突变有关,但不排除其他替加环素耐药机制的存在。

Objective To investigate the mechanism of resistance nodulation division(RND)efflux pumps mediated tigecycline-insensitive multidrug-resistant Acinetobacter baumannii(MDR-AB).Methods A total of 21 isolates of tigecycline-insensitive Acinetobacter baumannii and 39 isolates of tigecycline-sensitive MDR-AB in the Fourth Affiliated Hospital of Harbin Medical University from 2015 to 2018 were collected.Using broth microdilution method as the standard method,tigecycline-insensitive isolates[minimun inhibitory concentration(MIC)≥2μg/mL]were used as experimental group,and tigecycline-sensitive isolates(MIC≤1μg/mL)were used as control group.Polymerase chain reaction(PCR)was used to determine RND efflux pump genes for tigecycline sensitivity decreasing of Acinetobacter baumannii(adeB,adeG and adeJ)and upstream regulatory genes(adeS,baeR and baeS),and adeS amplification products were sequenced to find insertion sequence.Real-time quantitative polymerase chain reaction(RT-qPCR)was used to determine and compare the transcript levels of efflux pump adeABC,adeFGH,adeIJK,adeRS and baeRS in experimental and control groups.Results In the 21 isolates of Acinetobacter baumannii,12 isolates of tigecycline-insensitive MDR-AB were screened.The RND gene detection rate associated with decreased tigecycline sensitivity was 100%,and the ISAbaⅠinsertion mutation of adeS was found in the 2 isolates. The expression of adeABC in experimental group was higher than that in control group,which were 3.3,3.5 and 2.7 times of the standard isolate(ATCC 19606),respectively. The expression level ofadeFGH was increased,and the levels of adeIJK,adeRS and baeRS were slightly up-regulated in some isolates.Conclusions RND efflux pump mediated MDR-AB with decreased tigecycline sensitivity is related to highexpressions of adeABC and adeFGH. The high expression of adeABC is mainly related to the presence of ISAbalinsertion mutation in the upstream regulatory gene adeS,and there may be other tigecycline resistance mechanisms.