幽门螺杆菌耐甲硝唑基因分型与序列分析

Genotyping and sequencing of metronidazole resistance gene rdxA in Helicobacter pylori

目的 探讨幽门螺杆菌 (Helicobacterpylori,Hp)rdxA基因型及其与甲硝唑耐药性的关系及耐药的分子机制。方法 对 5 4例包括胃炎、消化性溃疡、胃癌患者的Hp分离株进行甲硝唑敏感实验 ,得到敏感株和耐药株 ;提取Hp基因组DNA ,PCR扩增rdxA基因 ,并进行限制性片段长度多态性(RFLP)分型 ;统计学分析基因型和耐药性关系 ;对 1株敏感株和 2株耐药株rdxA基因测序分析突变情况。结果 rdxA基因可分为两型 :Ⅰ型和Ⅱ型。Ⅰ型中的敏感株和耐药株所占比例为 5 6 %和44 %,Ⅱ型中的敏感株占 16 %,耐药株占 84%。经统计学分析 ,rdxA基因Ⅱ型与耐药性有相关性。rdxA基因测序分析显示耐药株存在编码区碱基置换及插入造成的移码突变 ;非编码区亦存在单个碱基缺失及置换突变。结论 rdxA基因Ⅱ型与Hp的甲硝唑耐药性密切相关 ,耐甲硝唑株主要表现为Ⅱ型。rdxA基因突变是耐甲硝唑的主要原因。

Objective To explore the relationship of rdxA genotype with the resistance to metronidazole(Mtz) in Helicobacter pylori(Hp), and molecular mechanism of the resistance. Methods Susceptibility tests were done for Hp isolates from 54 of cases including gastritis, ulcer and gastric cancer. Susceptable and resistant strains were respectively isolated. Chromosome DNA of the strains were extracted and rdxA genes were amplified by PCR. Genotyping of the gene was made by restrictive fragment length polymophism(RFLP). Relationship of the genotype to drug resistance was analysed by statistics. rdxA genes from one sensitive and two resistant strains were sequenced. Results There are two kinds of patterns called genotypesⅠand Ⅱ of rdxA gene in Hp. Sensitive and resistant strains are with rate of 56% and 44% respectively in typeⅠbut 16% and 84% in type Ⅱ. There is a significant relationship between the genotype Ⅱ and Mtz resistance (P<0.01). rdxA gene sequence shows that there are mutations of bases replacement and insertion, leading to frameshifting in coding region of rdxA in resistant strains. In non-coding region it shows mutation of single nucleotide deletion or substitution. Conclusion There is a significant relationship between rdxA genotype and Mtz resistance and the majority of resistant stranis are genotype Ⅱ. Mutation of rdxA gene is major reason of Mtz resistance.