| Background and objective: Helicobacter pylori(Hp) is a gastric pathogen that chronically infects more than half of the world's population. Infection with H. Pylori is strongly associated with chronic gastritis, peptic ulceration, gastric cancer and MALT Lymphoma. In 1994, Hp has been classified as a group I carcinogen by the International Agency for Research on Cancer (IARC). Multiple studies have demonstrated that eradication of Hp infection was essential. There were several antibiotics such as metronidazole, clarithromycin, amoxicillin and tetracycline in the method of eradication of H. Pylori. The resistance to these four antibiotics has developed and affected badly anti-H. Pylori therapies. The resistance to antibiotics was strongly associated with mutations in genes, for example, the resistacne to metronidazole was associated with mutation in rdxA gene; the resistacne to clarithromycin was associated with mutation in 23S rRNA; the resistacne to amoxicillin was associated with mutation in pbp1A; the resistacne to tetracycline was associated with mutation in 16S rRNA. Because of limit on technology, it is impossible to discover really the changes of the DNA sequence in genes previously. The aim of this study was to examine the difference between the resistance-strains and susceptive-strains by sequencing. Specifically, we wanted to examine the role of the mutation in gene to the development of resistance. Method: 262 Hp strains from gastric biopsy specimens were isolated on Hp selective medium (Skirrow's) containing 7–10% sheep defibrinated blood . Hp strains were cultured under microaerobic conditions at 37°C for 3–5 days, and then they were stored at ?80°C. Disk diffusion test was used to determine the susceptibility of 204 clinical Hp strains to antibiotics (metronidazole, clarithromycin, amoxicillin, tetracycline) . The segment of rdxA was amplified by PCR from MTZR strains , MTZS strains and Hp 11637. The segment of pbp1A was amplified by PCR from AMXR strains , AMXS strains and Hp 11637 by PCR. The segment of 23S rRNA was amplified by PCR from CLRR trains , CLRS strains and Hp 11637 by PCR. The segment of 16S rRNA was amplified by PCR from TETR trains , TETS strains and Hp 11637. These PCR-products were sequenced, and the nucleotide sequences were analyzed by the DNAStar software package and NCBI Blaster. In DNAstar and NCBI Blaster, these DNA sequences and aminophenol sequences aligned the standard strains respectively on the Genebank. Result: ①The rate of H. pylori resistance to metronidazole was 50.0%(102/204), to amoxicillin 4.4%(9/204), to clarithromycin 8.8%(18/204) and to tetracycline 4.4%(9/204).②The DNA homology of rdxA in MTZR and MTZS was 93.25%±1.93% and 93.79%±1.14% respectively compared with this gene in the standard strain 26695, P>0.05, but the aminophenol homology of RdxA in MTZR and MTZS was 67.02% ±22.80% and 82.61% ±6.83% respectively compared with the RdxA in the standard strain 26695, P<0.05. ③The DNA homology of pbp1a in AMXR and AMXS was 90.26%±3.45% and 95.52%±2.73% respectively compared with the gene in the standard strain 26695, P<0.01, and the aminophenol homology of PBP1A in AMXR and AMXS was 40.52%±18.48% and 93.28%±6.21% respectively compared with the PBP1A in the standard strain 26695, P<0.01. ④The DNA homology of 23S rRNA in CLRR and CLRS was 98.68%±0.54% and 98. 33%±0.96% respectively compared with the gene in the standard strain HpU27270, P>0.05. seven of the 17 CLRR strains carried the A2144G mutation, none of the CLRS 15 starins did, P<0.01. ⑤The DNA homology of 16S rRNA in TETR and TETS was 98.20%±0.55% and 98. 16%±0. 65% respectively compared with the gene in the standard strain ATCC43504, P>0.05. Five of the 9 TETR strains carried the AGA926-928TTC mutation, none of the TETS 11 starins did, P<0.01. Conclusion: ①The rate of H. pylori resistance to metronidazole was high,the resistance to clarithromycin, amoxicillin and tetracycline has arisen and the rate of them was low. ②Aminophenol mutation in RdxA caused by mutational rdxA was strongly associated with resistance to metronidazole. The form of mutation in rdxA included frameshift mutation and missense mutation, but it's location was still unclear. ③Aminophenol mutation in PBP caused by mutational pbp1A was strongly associated with resistance to amoxicillin. ④The mutation A2144G was associated with resistance to clarithromycin, and the other kinds of mutation of 23S rRNA were discovered in CLRR strains. ⑤The mutation AGA926-928TTC and analogical mutation were associated with resistance to tetracycline, and the other kinds of mutation of 16S rRNA were discovered in CLRR strains.
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