The Research of mechanisms for tetracycline and multi-drug resistant Helicobacter pylori
| Autor: | Jeng-Yih Wu, 吳政毅 |
|---|---|
| Rok vydání: | 2003 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 91 Background: Successful treatment of H. pylori infection heals or prevents the H. pylori related-diseases: gastric ulcer, duodenal ulcer, atrophic gastritis, gastric adenocarcinoma, and MALToma. The golden standard of eradication therapy for H. pylori is three-combined therapy, i.e. one proton pump inhibitor and two antibiotics. Amoxicillin, clarithromycin, metronidazole, and tetracycline are antibiotics most frequently used. However, the presence of antibiotic resistant H. pylori has become the major impediment preventing successful therapy. Although tetracycline resistant H. pylori have reported, little was known about the resistant mechanism of tetracycline resistant H. pylori. Tetracycline is a bacteriostatic antibiotic by means of binding with 16S rRNA and thus inhibition of protein synthesis. It is hypothesized that point mutations at the major binding site of gene encoding 16S rRNA could account for the development of tetracycline resistant H. pylori. The aim of this study are (1) to identify the prevalence rate of resistant H. pylori to frequently used antibiotics, (2) to find out if clinical tetracycline resistant H. pylori is present, (3) if so, to investigate the mechanism(s) of resistance and its characterizations, (4) to test their minimal inhibition concentration of other antibiotics, (5) to check the presence of multi-drug resistant H. pylori, (6) to study the mechanism of multi-drug resistant H. pylori. Materials and Methods: 413 clinical H. pylori isolates were obtained from gastric biopsies culture of 227 patients between 1999 and 2000. Agar dilution measurements were used for the determination of minimal inhibition concentration of antibiotics to these clinical isolates. Genomic DNA was extracted from these tetracycline resistant H. pylori isolates. PCR for tetracycline major binding site of 16S rRNA was undergone using designed primer and these genomic DNA. DNA sequences were analysed after PCR fragments were purified. RAPD-PCR was done to determine the strain pattern in these tetracycline resistant H. pylori isolates. Natural transformation was performed using the purified genomic DNA and PCR fragments. The ribosomal binding assay was also investigated among specific mutated strains. Intracellular tetracycline accumulation assay and outer membrane proteins of specific strains were checked if other mechanisms were involved in the resistance of tetracycline. Results: Thirteen tetracycline resistant H. pylori isolates were found in 413 clinical isolates. Among these 13 isolates, only 10 different patterns were found by RAPD-PCR. Four single point mutations were noted, ie. tGA, AGc, AGt, gGA in stead of AGA965-967 at tetracycline major binding site of 16S rRNA encoding gene. The ribosome purified from the mutated isolated showed decreased affinity to tetracycline compared with the ribosome from susceptible strain. Based on the M.I.C. test, 2 isolates with high resistance to tetracycline should be considered as multi-drug resistant H. pylori. Those 2 strains did not show mutations at tetracycline major binding site of 16S rRNA encoding gene. Decreased intracellular tetracycline accumulation which could be related with the change of outer membrane proteins may account for the development of resistance to tetracycline in these 2 strains. Conclusion and Discussion: From the data of decreased tetracycline affinity of ribosome from those mutated tetracycline resistant H. pylori isolates, the point mutations at the gene encoding tetracycline major binding site of 16S rRNA should be responsible for the development of tetracycline resistance. Another tetracycline resistant mechanism should be related with the change of outer membrane protein resulting to the decreased intracellular antibiotics accumulation. This is also an important mechanism for the multi-drug resistant H. pylori. |
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