鲍曼不动杆菌在环境美罗培南浓度变化时耐药性的改变及其机制  

Changes in drug resistance of Acinetobacter baumannii during the change of meropenem concentration in the environment and its mechanism

作　　者：赵富茂 彭玫 彭晓露 舒韦韦 彭丽[1] ZHAO Fumao;PENG Mei;PENG Xiaolu;SHU Weiwei;PENG Li(Department of Respiratory and Critical Care Medicine,The First Affiliated Hospital of Chongqing Medical University,Chongqing 400016,China;Department of Critical Care Medicine,Yongchuan Hospital of Chongqing Medical University,Chongqing 402160,China)

机构地区：[1]重庆医科大学附属第一医院呼吸与危重症医学科,重庆400016 [2]重庆医科大学附属永川医院重症医学科,重庆402160

出　　处：《上海交通大学学报（医学版）》2023年第11期1396-1407,共12页Journal of Shanghai Jiao tong University:Medical Science

基　　金：重庆市卫生健康委员会医学科研计划项目(2017ZDXM004);重庆市社会事业与民生保障科技创新专项项目(cstc2017shmsA130031)。

摘　　要：目的探寻鲍曼不动杆菌(Acinetobacter baumannii)在环境中碳青霉烯类药物美罗培南浓度改变时耐药性变化的机制。方法通过改变鲍曼不动杆菌标准敏感株ATCC19606和临床耐药株AB.2014培养环境中的美罗培南浓度等条件,诱导对美罗培南不同耐药程度的衍生株。测量所得菌株的生长曲线,并提取各菌株的DNA和RNA,采用PCR分析菌株耐药性改变后的碳青霉烯酶基因IMI、KPC、GES-1、IMP、VIM、NDM-1、OXA23、OXA24、OXA51、OXA58的表达情况;通过实时荧光定量PCR(real-time fluorescent quantitative PCR,RT-qPCR)分析不同耐药程度的鲍曼不动杆菌耐药基因,包括OXA51,外排泵基因adeB、adeG、adeJ,孔蛋白基因carO、omp33-36、oprC,青霉素结合蛋白基因ponA的表达水平变化;通过全基因组测序及生物信息学工具分析耐药性改变后菌株的差异基因富集情况的变化。结果获得了鲍曼不动杆菌ATCC19606与AB.2014对美罗培南不同耐药程度的11个衍生株,最低抑菌浓度(minimum inhibitory concentration,MIC)为1~128μg/mL。ATCC19606及其衍生株的生长速度和峰值随着耐药性的增加而降低,但AB.2014及其衍生株并没有表现出这种趋势。ATCC19606及其衍生株表达3个碳青霉烯酶基因OXA51、VIM和IMP,AB.2014及其多数衍生株表达4个碳青霉烯酶基因OXA23、OXA51、VIM和IMP,仅AB.2014的一个复敏衍生株出现了OXA23丢失。RT-qPCR结果显示,仅在ATCC19606及其耐药衍生株中oprC基因的表达量随着耐药性的升高而降低,多数耐药基因的表达水平与菌株的耐药水平变化一致。生物信息学分析提示ATCC19606不同衍生株之间的差异基因主要富集于铁载体摄取跨膜转运体活性、细胞外膜、细菌分泌系统和群体感应等,而AB.2014不同衍生株之间的差异基因主要富集于细胞外膜、细胞对化学刺激的反应、阿特拉津降解和RNA聚合酶等。结论碳青霉烯类药物环境压力会引起鲍曼不动杆菌耐药性�Objective To explore the mechanism of changes in resistance to meropenem(MEM),a carbapenem drug,in Acinetobacter baumannii(A.baumannii)cultured in different antibiotic concentrations.Methods Through changing the MEM concentration and other culture conditions of the standard sensitive strain of A.baumannii ATCC19606 and the clinical drugresistant strain AB.2014,the derived strains with different levels of MEM-resistance were induced.The growth curves of all the stains were detected.DNA and RNA of them were extracted.PCR was used to analyze the expression of carbapenemase genes,including IMI,KPC,GES-1,IMP,VIM,NDM-1,OXA23,OXA24,OXA51,and OXA58.Real-time fluorescent quantitative PCR(RTqPCR)was used to analyze the expression levels of the carbapenemase gene(OXA51),efflux pump genes(adeB,adeG,and adeJ),pore protein genes(carO,omp33-36,and oprC)and the penicillin-binding protein gene(ponA)in the A.baumannii strains with different resistance to MEM,of which the differential gene enrichment was also detected by whole genome sequencing and bioinformatics tools.Results The 11 derived strains of ATCC19606 and AB.2014 with different levels of resistance to MEM were obtained,of which the minimum inhibitory concentrations(MIC)were 1‒128μg/mL.The growth rates and peak values of ATCC19606 and its derivatives decreased with the increase of drug resistance,but AB.2014 and its derivatives did not show this trend.ATCC19606 and its derived strains expressed 3 carbapenemase genes,i.e.,OXA51,VIM and IMP,while AB.2014 and most of its derived strains expressed 4 carbapenemase genes,i.e.,OXA23,OXA51,VIM and IMP,with only one sensitized derivative of AB.2014 losing OXA23 gene.RT-qPCR results showed that only in ATCC19606 and its drug-resistant derivatives,the expression level of oprC gene decreased with the increase of drug resistance,and the expression levels of most drug-resistant genes were consistent with the changes of drug resistance levels of the strains.Bioinformatics analysis indicated that the differential genes among different

关 键 词：鲍曼不动杆菌 美罗培南 耐药性 碳青霉烯酶 外排泵 孔蛋白 青霉素结合蛋白 

分 类 号：R378.99[医药卫生—病原生物学]