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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 analyze the molecular mechanism of amoxicillin resistance in Helicobacter pylori (Hp). Methods Hp was inoculated continuously on Karmali plates containing amoxicillin, and the drug resistance was induced in Hp 26695, a sensitive strain of amoxicillin. The resistant strains were obtained. The coding genes of five penicillin-binding proteins (PBPs, PBP1-PBP5), two Hop-like proteins (HopB and HopC) and one efflux pump protein (HefA) were amplified by PCR. Gene differences between. Results Four strains of amoxicillin were obtained. The MICs of the two strains were 0.5 and 2 mg / L, and the MIC was 4 mg / L. The DNA fragments amplified by PCR were sequenced and compared Three mutations, two substitution mutations (T438M, T593K) and one insertion mutation (594G +) were detected in the PBP1 of the four drug-resistant strains. The amino acid changes caused by the mutations were all located in the C End transpeptidase domain. T593K and 594G + are newly discovered mutation sites. The MIC for the 594G + mutant was 2.0 mg / L. T438M and T593K mutations increased the drug resistance of 594G + mutant, and the MIC of 594G + combined with T438M or T593K mutant increased to 4.0 mg / L. Conclusion Three mutations were detected in Hp PBP1 induced by amoxicillin in vitro, of which two were newly found. The resistance of Hp to amoxicillin might be related to the above mutations of PBP1, but further experiments were needed .