Study On Resistance Condition Of Helicobacter Pylori And Mechanism Of Post-treatment Resistance

Background and Objective:Helicobacter pylori (H.pylori) is a Gram-negative, microaerophilic, spiral bacterium that colonises the stomachs of approximately half the world's population. Infection with H.pylori is associated with chronic gastritis and peptic ulceration and the bacterium is also considered a risk factor for the development of gastric adenocarcinoma and mucosa-associated lymphoid tissue (MALT) lymphoma. Currently the most effective treatment regimens for H.pylori combine a proton pump inhibitor and two antimicrobial agents selected from amoxicillin, metronidazole, or clarithromycin. Although optimal first-line therapy is associated with high cure rates, the rising prevalence of resistance to antibiotic component of current eradication regimens increasingly threatens to compromise the efficacy of these regimens. Recently, there have been considerable researches in unraveling the factors that determine antibiotic susceptibility in H.pylori. These researches comfirm that the resistance to antibiorics was associated with mutation in related gene, 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. But most of these researches were based on primary resistance strains or in vitro induced resistance stains, so it is difficult to interpret the emergence of resistance after treatment failure. The aim of this study was to investigate the changes of antibiotics resistance rates in recent 3 years, to compare the efficacy of three treatment regimens, to identify the difference by gene sequence among the primary susceptive-strains, primary resistance-strains and post-treatment resistance-strains; and to explore the role of the gene mutation in the development of resistance.Method:Two gastric biopsy specimens were taken from patients with digestive symptoms by endoscopic examination ,whose rapid urease enzyme test was positive. Each specimen was cultured on selective medium(Skirrow's) containing 5-10% sheep defibrinated blood. Hp strains were cultured under microaerobic condition at 37℃for 3–5 days, and then stored at ?80℃. A total of 345 Hp strains with no treatment before were isolated and stored. Disk diffusion test was used to determine the susceptibility of clinical Hp strains to antibiotics. A total of 79/345 H.pylori-positive patients were randomly enrolled to received one of the three follow 7-day regimens. GroupA: PPI, AMX, CLA; GroupB: PPI, CLA, MTZ; GroupC: PPI, CBS, TI, CLA. Bacterial eradication was assessed by C14-urea breath test 4 weeks after the therapy had ended. There are 15 patients with H.pylori-positive. Additionally, we have got another 18 patients with eradication failure(with no Hp culture and antibiotic susceptibility test before eradication treatment). A total of 20/33 H.pylori strain have been isolated and disk diffusion test was used to determine the antibiotic susceptibility . The rdxA gene fragment was amplified by PCR from MTZR strains and MTZS strains. The pbp1A gene fragment was amplified by PCR from AMXR strains and AMXS strains . The 23S rRNA gene fragment was amplified by PCR from CLRR trains and CLRS strains. PCR-products were sequenced, and the nucleotide sequences were analyzed by the DNAStar software package. These DNA sequences and aminophenol sequences were compared with the standard strain from the Genebank respectively to analyse the difference between them.Result:①I n 2004,2005,2006, the rate of H.pylori resistance to metronidazole was 50.0%, 55.6%, 53.1% respectively, p>0.05; the rate of H.pylori resistance to amoxicillin was 4.4%, 8.9%, 10.4% respectively, p>0.05; the rate of H.pylori resistance to clarithromycin was 8.8%, 11.1%, 9.4% respectively, p>0.05; the rate of H.pylori resistance to tetracycline was 4.4%, 4.4%, 5.2% respectively, p>0.05. The total rate of H. pylori resistance to metronidazole was 51.6%, to amoxicillin 6.7%, to clarithromycin 9.3% and to tetracycline 4.6%. p<0.05.②The eradication rate in all 79 patients who had underwent susceptibility testing before the eradication therapy was 81.0%(64/79). The eradication rates for each group were 87.8(36/41) in group A,72.7%(16/22) in group B, 75%(12/16) in the group C, respectively with no significant differences.③The eradication rates of H.pylori strains with one antibiotic resistance and two antibiotics resistance were 61.1%(11/18) and 0(0/5), respectively. The eradication rate of sensitive H.pylori strains was 94.6%(53/56). The eradication rates were significantly different between the resistant and sensitive strains of H.pylori , p<0.01. The eradication rates were significantly different between the one antibiotic resistant strains and two antibiotics resistance strains , p<0.05.④The rate of H.pylori resistance to metronidazole, amoxicillin and clarithromycin after treatment were 85% 25%, 50%, respectively, p<0.05.⑤Comparing the rdxA gene of primary MTZS strains, primary MTZR strains and post-treatment MTZR strains with standard strains, we found DNA homology of all the clinical strains are high, p>0.05. Aminophenol homology of the primary MTZR was lower than primary MTZS and MTZR,p<0.05.⑥Comparing the pbp1A gene of primary AMXS strains, primary AMXR strains and post-treatment AMXR strains with standard strain, we found DNA homology of all the clinical strains are high, p>0.05. Aminophenol homology of AMXR strains was lower than AMXS strains, p<0.05.⑦Comparing the 23S rRNA gene of primary CLAS strains, primary CLAR strains and post-treatment CLAR strains with standard strain, we found DNA homology of all the clinical strains are high, p>0.05. And we found A2144G (7/17)mutation in primary CLAR strains, A2144T (5/8)mutation in post-treatment CLAR strains; but no mutation was found in 2144 position(0/15) of CLAS strains, p<0.05. We also found G2255T(8/8), G2173T(7/8) and T2183C(7/8) mutation in post-treatment CLAR strains, these point mutation were not found in primary CLAR (0/17)and CLAS (0/15)strains, p<0.01.Conclusion:①In the recent 3 years, the rate of H.pylori resistance to metronidazole, amoxicillin, clarithromycin and tetracycline had no significant change. The rate of resistance to metronidazole was higher than the other antibiotics.②antibiotics resistance is the main factor which influences the eradication rate of H.pylori.③These three common used eradication regimens based on PPI have similar eradication rates of H.pylori.④The rate of resistance to metronidazole was high after treatment failure.⑤rdxA gene mutation may be the main reason of primary MTZR, but there might be some other reason on the generation of resistance in post-treatment MTZR H.pylori.④Aminophenol mutation in PBP caused by mutational pbp1A was associated with resistance to amoxicillin.⑤T he mutation in position 2144 was associated with resistance to clarithromycin, and some new point mutations G2255T, G2173T in 23S rRNA were discovered in CLRR strains.