贵阳地区351株幽门螺杆菌药物敏感性及pbp1多样性分析

Drug resistance and diversity of pbp1 of local Helicobacter pylori isolates obtained in Guiyang city, China

目的了解贵阳地区幽门螺杆菌(Helicobacter pylori,H.pylori)对5种抗生素药物敏感性及阿莫西林耐药菌株pbp1突变特征。方法采用界值法检测351株幽门螺杆菌临床菌株对5种抗菌药物的敏感性,对各药物耐药率以χ^2检验进行比较。选取20株阿莫西林耐药菌株及相等数量的敏感菌株,提取基因组DNA,PCR扩增pbp1基因功能区并测序,采用DNAStar软件将核苷酸序列转换为氨基酸序列后进行比对和分析。结果351株幽门螺杆菌临床菌株对甲硝唑、克拉霉素、阿莫西林、左氧氟沙星和四环素5种药物的耐药率分别为71.56%、34.60%、13.27%、40.28%、17.06%。20株阿莫西林耐药和20株敏感菌株PBP1氨基酸序列分析结果显示,PBP1多样性复杂,在耐药菌株特有变异位点中,PBP1氨基酸的S543R变异频率最高,为75%,其次是N641D为60%。结论本研究中的幽门螺杆菌对5种抗生素均有不同程度耐药,其中阿莫西林和四环素耐药率较其他地区高,并且双重耐药与多重耐药在总体耐药中所占比例较大;阿莫西林耐药菌株PBP1氨基酸存在多位点多态性,可能与幽门螺杆菌阿莫西林耐药有关。

The aim of this study is to determine the susceptibility of Helicobacter pylori to five kinds of antibiotics and to analyze the diversity of pbp 1 in amoxicillin-resistant strains found in Guiyang city, China. The boundary value method was used to determine susceptibility of 351 clinical strains of Helicobacter pylori to five antimicrobial agents. A χ^2 test was used to analyze drug resistance rates. Genomic DNA was extracted from 20 amoxicillin-resistant strains and an equal number of amoxicillin-sensitive strains. The functional regions of pbp1 were ampli fied by PCR and sequenced, and the sequencing results were analyzed by DNAStar software. The drug resistance rates observed in 351strains of Helicobacter pylori isolates to metronidazole, clarithromycin, amoxicillin, levofloxacin, and tetracycline were 71.56%,34.60%,13.27%,40.28%,17.06%, respectively. Amino acid sequence of PBP1of these 20 amoxicillin-resistant and 20 amoxicillin-sensitive strains showed complex diversity. Among the unique mutations observed in drug-resistant strains, the most frequent mutation of PBP1 resulted in S543R (75%), while the second most frequent mutation resulted in N641D (60%). Helicobacter pylori in this study was resistant to five antibiotics to varying degrees, and among these, amoxicillin and tetracycline resistance rates were higher than other areas in China. In addition,dual and multiple drug resistance accounted for a large proportion of total drug resistance. Multi-site mutations observed inPBP1 derived from the amoxicillin-resistant strains may be related to resistance of H. pylori to amoxicillin.