体外诱导阿莫西林耐药株幽门螺杆菌PBP1突变位点检测  

New mutations of PBP1 in amoxicillin-resistant strains of Helicobacterpyloriinduced in the laboratory

作　　者：曹奇志[1,2] 何利华[1] 宫雅楠[1] 张建中[1] 

机构地区：[1]中闰疾病预防控制中心传染病预防控制所传染病预防控制国家重点实验室,北京102206 [2]滨州医学院免疫教研室,山东烟台264003

出　　处：《中国病原生物学杂志》2015年第1期6-8,16,共4页Journal of Pathogen Biology

基　　金：“十二五”国家科技支撑计划项目(No.2012BAI06B02)

摘　　要：目的分析幽门螺杆菌（Hp）阿莫西林耐药的相关分子机制。方法将Hp接种在含阿莫西林Karmali平板连续传代,诱导阿莫西林敏感菌株Hp 26695产生耐药性,获得耐药株。PCR扩增5种青霉素结合蛋白（PBPs,PBP1~PBP5）,2种Hop孔蛋白（HopB和HopC）和1个外排泵蛋白（HefA）的编码基因,分析耐药菌株与敏感株Hp 26695之间的基因差异。结果获得4株阿莫西林耐药菌株,其中最小抑菌浓度（MIC）为0.5和2 mg/L各1株,MIC为4mg/L 2株;对PCR扩增的基因片段进行测序和比对分析在4个耐药株的PBP1中共检出3个突变位点,2个替代突变（T438M,T593K）和1个插入突变（594G＋）,突变导致的氨基酸变化均位于所有耐药株PBP1的C端转肽酶结构域中。T593K和594G＋是新发现的突变位点。594G＋突变株的MIC为2.0mg/L。T438M和T593K突变增加了594G＋突变株的耐药强度,594G＋合并T438M或T593K突变株的MIC提高到4.0mg/L。结论在体外诱导的阿莫西林耐药株Hp PBP1中检测到3个突变位点,其中2个为新发现突变,Hp对阿莫西林耐药可能与PBP1的上述突变有关,但需要进一步试验验证。Objective To elucidate the molecular mechanisms involved in Helicobacter pylori resistance to amoxicillin.Methods Amoxicillin-resistant isolates were obtained from the susceptible strain H.pylori 26 695 by serial passaging on Karmali plates containing amoxicillin.Sequencing of five PBPs（PBP1-5）genes,two Hop porin genes（hopB and hopC）,and one efflux pump gene（hefA）of resistant isolates and ATCC 700392 was performed. Results Four amoxicillin-resistant isolates were obtained.The MIC of the 4isolates was 0.5,2.0,4.0,and 4.0mg/L,respectively.Three mutations（T438M,T593 K,and 594G＋）of PBP1 were detected only in amoxicillin-resistant isolates.All of the mutations were around the second or the third penicillin-binding motif located in the transpeptidase region of PBP1.This study noted the new mutations of T593 Kand 594G＋.594G＋conferred a high level resistance to amoxicillin（MIC：2.0mg/L）.In PBP1,the combination of 594G＋and T438 M or T593 Kconferred a higher level of resistance to amoxicillin（MIC：4.0mg/L）than the 594G＋mutation alone. Conclusion This study noted three mutations of PBP1 in amoxicillin-resistant isolates,including two novel mutations.These mutations may be involved in amoxicillin resistance.No mutations were noted in seven other genes.

关 键 词：幽门螺杆菌 阿莫西林 耐药性 突变 青霉素结合蛋白 

分 类 号：R378.91[医药卫生—病原生物学]