四環黴素與多重葯物抗藥性幽門螺旋桿菌抗葯機轉之研究

The Research of mechanisms for tetracycline and multi-drug resistant Helicobacter pylori

吳政毅(Jeng-Yih Wu)

指導教授 ： 王文明

高雄醫學大學/醫學院/醫學研究所/碩士(2003年)

摘要

前言：與胃幽門螺旋桿菌相關的上消化道疾病，如胃潰瘍、十二指腸潰瘍、萎縮性胃炎、胃癌、胃黏膜相關淋巴組織淋巴瘤，可以經由成功的根除胃幽門螺旋桿菌而得到治療與預防已是眾所週知不爭的事實。目前，根除療法的黃金準則是三合一療法；亦即一種氫離子阻斷劑加上兩種抗生素。常用的抗生素有amoxicillin, clarithromycin, metronidazole,與tetracycline。然而胃幽門螺旋桿菌根除療法無法成功的主要原因之一，即是抗藥性菌株的不斷出現。雖然已有一些抗四環黴素抗藥性幽門螺旋桿菌的報告，但對於抗四環黴素幽門螺旋桿菌的抗藥性機轉的研究尚未十分明瞭。四環黴素的葯理機轉主要是跟細胞的核醣體，特別是16S核醣體核醣核酸結合，進而抑制細胞蛋白質的合成，而達到制菌的效果。因此吾人假設：轉譯16S核醣體核醣核酸的去氧核醣核酸產生點狀突變，可能與抗四環黴素幽門螺旋桿菌的抗藥性產生有關。本篇研究的主要目的是研究：（1）對常用抗生素有抗藥性的胃幽門螺旋桿菌盛行率，（2）並試圖找出抗四環黴素幽門螺旋桿菌的臨床菌株，（3）觀察抗四環黴素幽門螺旋桿菌的特徵與機轉之研究，（4）及其對其他常用抗生素的最小抑菌濃度，（5）是否有多重抗藥性幽門螺旋桿菌的出現，（6）多重抗藥性幽門螺旋桿菌臨床菌株的特徵與機轉之研究。
材料與方法：自西元1999年到西元2000年間，從227位病人經胃內試鏡切片組織培養所得到的413株幽門螺旋桿菌臨床菌株中進行藥物敏感性之相關研究。在最小抑菌濃度的測量方面，是採用瓊脂稀釋法，並進一步純化所得的抗四環黴素幽門螺旋桿菌菌株的去氧核醣核酸，利用此一純化的去氧核醣核酸與所設計的引子進行多重聚合脢連鎖反應，將所得的多重聚合脢連鎖反應片段加以純化，並分析轉譯16S核醣體核醣核酸的去氧核醣核酸可能產生的點狀突變，並利用隨機放大多形態去氧核醣核酸多重聚合脢連鎖反應(RAPD-PCR)分析所得菌株的去氧核醣核酸形態，同時利用所得的純化去氧核醣核酸與多重聚合脢連鎖反應片段對敏感性菌株做自然轉型，並分離抗四環黴素幽門螺旋桿菌的核醣體與氚標記之四環黴素進行核醣體--四環黴素結合力測定，也進行四環黴素細胞內累積測定，並純化這些菌株之外膜蛋白加以分析。
結果：413株幽門螺旋桿菌臨床菌株中，發現13株抗四環黴素幽門螺旋桿菌菌株。利用RAPD-PCR分析這13株抗四環黴素幽門螺旋桿菌菌株僅得到10種不同的形態。對轉錄四環黴素的主要結合位置16S核醣體核醣核酸的去氧核醣核酸進行分析，發現有4種單點突變（亦即tGA, AGc, AGt, gGA）。而這些單點突變菌株的核醣體--四環黴素結合力與敏感性菌株的核醣體--四環黴素結合力比較，有下降之情形。另有2株具有極高之抗四環黴素抗藥性菌株（應屬於多重抗藥性菌株），但此2株轉錄四環黴素的主要結合位置16S核醣體核醣核酸的去氧核醣核酸並無突變，而且細胞內四環黴素累積測定呈現下降，其抗藥性的產生應與外膜蛋白的變異有關。
結論與討論：經由這些單點突變菌株的核醣體與四環黴素結合力下降之情形，可說明在與四環黴素主要結合位置的16S核醣體核醣核酸的轉錄去氧核醣核酸突變，應是部分抗四環黴素幽門螺旋桿菌菌株之抗葯機轉，另一機轉與外膜蛋白的改變，導致藥物進入細胞的濃度降低有關。而後者也是多重抗藥性幽門螺旋桿菌的主要機轉之一。

關鍵字

幽門螺旋桿菌 ； 四環黴素 ； 核醣體 ； 多重抗藥性 ； 外膜蛋白

並列摘要

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.

並列關鍵字

Helicobacter pylori ； tetracycline ； ribosome ； multi-drug resistance ； outer membrane protein

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