Okamoto Takeshi, Yoshiyama Hironori, Nakazawa Teruko, Park In-Dal, Chang Myung-Woong, Yanai Hideo, Okita Kiwamu, Shirai Mutsunori
Department of Microbiology, Yamaguchi University School of Medicine, Minamikogushi 1-1-1, Ube, Yamaguchi 755-8505, Japan.
J Antimicrob Chemother. 2002 Dec;50(6):849-56. doi: 10.1093/jac/dkf140.
Reports on the isolation of amoxicillin-resistant Helicobacter pylori are increasing worldwide, which may cause serious problems in eradication therapy. To elucidate the mechanism of amoxicillin resistance of H. pylori, penicillin-binding proteins (PBPs) of amoxicillin-resistant strains isolated in Korea were analysed. Three PBPs (66, 63 and 60 kDa) were identified in both amoxicillin-resistant and -susceptible strains using biotinylated ampicillin, and the PBP profiles were very similar irrespective of the difference in amoxicillin susceptibility. We obtained clones with moderate resistance from an amoxicillin-susceptible strain, HPK5, by transformation with genomic DNA from an amoxicillin-resistant strain, HPA116. In a resistance-induced clone, HPO1, the affinity of PBP1 for amoxicillin was reduced. The pbp1 genes from HPA116, HPO1 and HPK5 were cloned and sequenced. The nucleotide sequences of pbp1 from HPA116 and HPO1 were almost identical, whereas that of HPK5 was quite different. Both the ORFs of HPA116 and HPO1 pbp1 have four substitutions and one insertion of amino acid residues compared with those of HPK5 and other sensitive strains. All the mutations, except one, are in the C-terminal half of the 659-amino-acid sequence containing the penicillin-binding modules. DNA fragments containing either full-length or a C-terminal half of pbp1 could transform HPK5 to have resistance, indicating that changes in the penicillin-binding core of PBP1 are involved in the amoxicillin resistance of H. pylori isolated in Korea.
全球范围内，关于耐阿莫西林幽门螺杆菌分离的报道日益增多，这可能在根除治疗中引发严重问题。为阐明幽门螺杆菌对阿莫西林耐药的机制，对韩国分离出的耐阿莫西林菌株的青霉素结合蛋白（PBPs）进行了分析。使用生物素化氨苄西林在耐阿莫西林和敏感菌株中均鉴定出三种PBPs（66、63和60 kDa），且无论阿莫西林敏感性如何差异，PBP图谱都非常相似。我们通过用耐阿莫西林菌株HPA116的基因组DNA转化耐阿莫西林敏感菌株HPK5，获得了具有中度耐药性的克隆。在耐药诱导克隆HPO1中，PBP1对阿莫西林的亲和力降低。对HPA116、HPO1和HPK5的pbp1基因进行了克隆和测序。HPA116和HPO1的pbp1核苷酸序列几乎相同，而HPK5的则有很大差异。与HPK5和其他敏感菌株相比，HPA116和HPO1 pbp1的开放阅读框均有四个氨基酸残基替代和一个插入。除一个突变外所有突变均位于包含青霉素结合模块的659个氨基酸序列的C端一半。包含pbp1全长或C端一半 的DNA片段均可将HPK5转化为具有耐药性，这表明PBP1青霉素结合核心的变化与韩国分离出的幽门螺杆菌对阿莫西林的耐药性有关。
