摘要
目的探讨阿莫西林(AMX)不稳定耐药的幽门螺杆菌(Hp)演化成AMX稳定高水平耐药的表型及其突变基因的检测。方法以冻存后的Hp菌株H390作为出发菌株,在不断增加AMX浓度的培养基上连续传代,筛选对AMX耐药的克隆,检测耐药克隆的最小抑菌浓度(MIC),置于-80℃冻存3个月后再复苏,根据冻存后MIC下降情况判断其耐药性是否稳定。对获得的AMX最高MIC值的克隆H390r和出发菌株H390进行基因组测序分析和外排泵抑制试验,检测并鉴定与H390r获得的AMX高水平耐药性相关的基因突变。结果通过AMX筛选获得4个AMX高水平耐药克隆,MIC分别为12、32、64和≥256 mg/L,经-80℃冻存后,MIC均未发生改变。相比于亲本菌株H390,AMX稳定耐药克隆H390r存在多个基因的突变,包括与AMX耐药性相关的编码RND外排系统的hefC、编码孔蛋白的hopB与hopC和编码青霉素结合蛋白的ftsI。H390r在有外排泵抑制剂存在时对AMX的MIC大幅降低。结论AMX能够在不稳定耐药的Hp中筛选出稳定耐药的克隆;H390r存在与AMX耐药相关的hefC、hopB、hopC和ftsI基因突变。这些突变可能是H390r获得AMX稳定高水平耐药的主要原因。
Objective To investigate the phenotype of amoxicillin(AMX)unstable resistant Helicobacter pylori(Hp)evolving into AMX stable high level resistance and the detection of its mutated genes.Methods Using the frozen Hp strain H390 as the starting strain,the clones resistant to AMX were continuously cultured on the medium with increasing AMX concentration,and the minimum inhibitory concentration(MIC)of the resistant clones was detected.After frozen at-80℃for 3 months,the drug resistance was stable according to whether the MIC decreased after frozen storage.Genome sequencing analysis and efflux pump inhibition assay were performed on cloned H390r and parental strain H390 with the highest AMX MIC value,and gene mutations associated with the high level AMX resistance obtained by H390r were detected and identified.Results Four AMX high level resistant clones were obtained by AMX screening with MICs of 12,32,64 and≥256 mg/L,respectively,and none of the MICs were altered after freezing at-80℃.Compared to the parental strain H390,the AMX stable resistant clone H390r had mutations in several genes,including hefC encoding the RND efflux system,hopB and hopC encoding the pore proteins and ftsI encoding the penicillin binding protein,which were associated with AMX resistance.H390r was substantially reduced in MIC to AMX in the presence of efflux pump inhibitors.Conclusion AMX can screen stable resistant clones from unstable resistant Hp.H390r had mutations in hefC,hopB,hopC,and ftsI associated with AMX resistance.These mutations may be the main reason why H390r acquired a stable high level of resistance to AMX.
作者
陆秋丹
糜孟衡
崔古贞
张峥嵘
吴晓娟
陈峥宏
Lu Qiudan;Mi Mengheng;Cui Guzhen;Zhang Zhengrong;Wu Xiaojuan;Chen Zhenghong(Key Laboratory of Microbiology and Parasitology of Education Department of Guizhou,School of Basic Medical Science,Guizhou Medical University,Guiyang 550025;Dept of Laboratory,Qiandongnan Prefecture People′s Hospital,Kaili 556000;Key Laboratory of Endemic and Ethnic Diseases,Ministry of Education,Key Laboratory of Medical Molecular Biology of Guizhou Province,Guizhou Medical University,Guiyang 550004)
出处
《安徽医科大学学报》
CAS
北大核心
2024年第1期39-44,共6页
Acta Universitatis Medicinalis Anhui
基金
国家自然科学基金(编号:81860353)
贵州医科大学国基培育项目(编号:20NSP005)
贵州省教育厅滚动支持省属高校科研平台团队项目(编号:黔教技〔2022〕019号)。
关键词
幽门螺杆菌
阿莫西林
不稳定耐药
稳定耐药
Helicobacter pylori
amoxicillin
unstable resistance
stable resistance
