【作者】 斯庆图娜拉；

【导师】 王江滨；

【作者基本信息】 吉林大学 ， 内科学， 2011， 博士

【摘要】 由于近年来人们已经认识到幽门螺杆菌(Helicobacter pylori, HP)感染与慢性胃炎、消化性溃疡、胃癌、胃淋巴瘤甚至还与多种胃肠外疾病的发生密切相关,所以根除HP治疗也就成为治疗或预防上述疾病的重要手段。但是含两种抗生素的三联或四联疗法根除方案的广泛联合应用不仅对人体具有毒副作用,扰乱胃肠道的生态平衡,而且还可诱导新的耐药菌株的出现,引发人群耐药性增加等社会问题。显然在人群中广泛使用抗生素治疗HP感染并非是根除HP的最佳方案,因此希望能成功地推出一种既有治疗、预防效能,又不增加人群耐药性的微生态疗法,就成为当今国际范围内该研究领域的热点问题。HP虽经消化道传播,但其在人群中的易感性存在很大差异,而且同为HP感染者,所患慢性胃病的临床表现也存在很大差异,对治疗的反应也不同,因此人们推论胃粘膜局部对外源性微生物的抵抗力以及生理状态下共栖菌抵抗外源性微生物的能力都可能与HP的致病性以及疾病的转归密切相关。随着微生态学的发展,人们开始关注是否可利用益生菌提高防护屏障的作用,干扰致病菌粘附于胃粘膜上皮细胞的能力,并试图利用益生菌免疫屏障作用拮抗病原菌的致病性,甚至达到清除病原菌的作用。但是到目前为止国内外关于益生菌对HP的拮抗作用还只局限于标准HP菌株,还尚未有人关注益生菌对已经发生抗生素耐药的HP是否也具有抑制效应,是否有助于HP耐药菌株感染的根除。本研究对不同慢性胃病患者胃粘膜中分离培养出的Hp菌株进行耐药分析,并筛选出Hp耐药菌株,进一步扩增了其耐药基因片段,并对该片段进行测序分析。此外还观察了本研究室预先已筛选到的抗HP嗜酸乳杆菌是否对于临床HP耐药菌株和体外诱导耐药的HP标准菌株也具有拮抗作用。主要结果如下：1、不同慢性胃病来源HP耐药特征及耐药变异规律分析从84例不同慢性胃病患者胃粘膜中分离培养出HP菌株,分析其对甲硝唑、克拉霉素和阿莫西林等临床常用一线根除方案中3种抗生素的耐药率,并分别从HP耐药菌株中扩增其耐药基因片段,通过测序分析,探讨其耐药变异规律。结果发现84例HP临床分离株对甲硝唑、克拉霉素和阿莫西林的耐药率分别为67.9%(57/84)、17.9%(15/84)和1.2%(1/84)其中10例对甲硝唑和克拉霉素混合耐药,1例对甲硝唑、克拉霉素和阿莫西林混合耐药。分析甲硝唑耐药HP菌株的rdxA DNA序列并与标准HP26695菌株的对应DNA序列进行比较,结果发现68个rdxA突变位点,但突变缺乏明显的规律,也未发现新的突变位点。克拉霉素耐药HP菌株的23S rRNA序列分析结果显示,2144位点的突变是克拉霉素耐药突变菌株的主要突变热点,突变的形式以碱基替代为主,而插入和缺失较少。7株出现2144位点A→G的替代,3株出现2289位点C→T替代,2株出现2267位点插入T。1例阿莫西林临床耐药菌株pbp1A基因序列分析结果显示,该基因存在多种形式的变异,但并无确切位点上的变化规律,与国际上通用HP26695菌株之间同源性为90%。2、体外诱导HP耐药菌株耐药变异规律分析以不同浓度的抗生素对HPSS1和HP11637标准HP菌株进行诱导,结果仅诱导出甲硝唑耐药株,未诱导出克拉霉素及阿莫西林耐药株,基因序列分析表明甲硝唑耐药基因突变发生在rdxA基因7320位点和7413位点,以碱基替换形式为主；标准HP菌株发生耐药后伴有形态学上的改变及尿素酶活性的下降。3、抗HP嗜酸乳杆菌对不同慢性胃病来源HP耐药菌株的抑制作用分析将抗HP嗜酸乳杆菌上清液分别在固体和液体培养条件下与HP临床耐药菌株和甲硝唑诱导耐药HP标准菌株共同进行培养,结果显示在固体培养条件下抗HP嗜酸乳杆菌上清液对抗生素耐药HP菌株具有明显的抑菌作用,在液体培养条件下抗HP嗜酸乳杆菌菌液能抑制抗生素耐药HP菌株的增殖和尿素酶活性。结论：1.84例不同慢性胃病来源HP临床分离株对甲硝唑、克拉霉素和阿莫西林的耐药率分别为67.9%(57/84)、17.9%(15/84)和1.2%(1/84)2.从57例甲硝唑临床耐药HP菌株扩增出rdxA基因的主要突变形式为碱基的替代、插入和缺失,但无规律的变异位点。从15例克拉霉素临床耐药菌株中扩增出的23 S rRNA基因的变异主要发生在2144位点。从1例阿莫西林临床耐药菌株扩增出的pbp1A基因序列表现了多种形式的基因变异,也无确切位点上的变化规律。3.从体外诱导的HP标准菌株中也可扩增至(?)rdxA耐药基因,其突变形式以碱基替换为主,同时伴有HP形态及尿素酶活性的变化。4.在固体和液体培养条件下分别证实本研究室预先已筛选到的抗HP嗜酸乳杆菌对多株HP临床耐药菌株以及甲硝唑诱导耐药HP标准菌株均产生明显的抑制效应,并明显抑制抗生素耐药HP菌株尿素酶活性。更多还原

【Abstract】 Objective:To analyze the drug-resisitance of HP strains achecied from patients with gastic diseases to different antibiotics by selecting the antibiotics-resistant strains and amplifing and sequencing the fragments of HP resistance gene in Jilin Province. And discuss the mechanism of drug-resistant.Methods:84 HP strains were achieved from patients and tested drug sensitive test to metronidazole, clarithromycin and amoxicillin by E-test to select the drug-resistant HP strains whose resistant gene fragments were amplified and sequenced.At the same time, HP standard strains,HP11637 and HPSS1,were tested antibiotics induced resistance in vitro. Then sequence the resistant gene of HP strains which appeared antibiotics-induced-resistant to metronidazole, clarithromycin and amoxicillin. And analyze mechanisms by which HP strains appeared antibiotics-resistant.Results:84 HP strains were acheived from patients and tested drug sensitive to metronidazole, clarithromycin and amoxicillin by E-test.The resistant rates were separately 67.9%(57/84)、17.9%(15/84) and 1.2% (1/84),in which 10 strains showed mixed drug resistant to metronidazole and clarithromycin, and 1 HP strain was mixed-resistant to metronidazole, clarithromycin and amoxicillin. HPSS1 and HP11637 strains were only introduced resistance to metronidazole in vitro. By comparing rdxA DNA sequence 7296-7815 of metronidazole resistant HP strains with the relative sequence of standard HP26695 strain,we found 68 mutation sites in rdxA and the mutations were lack of significant law,in which there were no obvious mutation hot spots.And by comparing with foreign literatures,there were no new mutation site. Collect and purify the amplified product by PCR and sequence it.Compare 23S rRNA sequence 2106-2320 of clarithromycin resistant HP strains with the corresponding sequences of standard strains HP26695. The results showed that 2144 site is one of the hot mutants in the clarithromycin resistance mutations.The mutantions were mostly in the form of base substitution,and fewer insertions and deletions happened. A→G in 2144 site was most frequent, which was found in seven HP strains, in three HP strains mutation C→T happened in 2289 site, T insertion in 2267 site happened in two HP strains.Compare with the forgein literatures and no new mutations sites were found in this study. By comparing pbp1A gene sequence from one amoxicillin-resistant strain with that from HP 26695, the results showed that homology was 90%.Conclusion:.The resistant rates of 84 HP strains acheived from patients to metronidazole, clarithromycin and amoxicillin were separately 67.9% (57/84),17.9%(15/84) and 1.2%(1/84).The result indicated that in Jilin province, HP clinical isolates were most resistant to metronidazole, followed by clarithromycin and amoxicillin less. By amplifing resistant gene fragments from drug-resistant strains, we found that mutations in rdxA geng of metronidazole resistant HP strains were mostly in the form of spot mutation, including the base of the substitution, insertion and deletion mutation without any law.2144 point mutation of Clarithromycin resistant strains in 23 S rRNA happened most frequently.Mutations in 2289,2267,2318 and other sites could also be found.However,no new mutation was found. Homology between one amoxicillin-resistant strain and HP 26695was 90%. Objective:To observe the inhibitory effects of anti-HP lactobacillus acidophilus (LA4 and LA6) on HP strains isolated from clinical patients and induced drug-resistant HP standard strains,and discuss the possibility in using anti-HP lactobacillus acidophilus to assitant with estabilishing antibiotic-resistant HP and preventing from HP re-infection.Methods:Add anti-HP Lactobacillus acidophilus (LA4 and LA6) supernatant to HP strain holes in solid plates,then microaerophilic culture 72 h in 37℃and record inhibition zone. Anti-HP bacterium Lactobacillus acidophilus were also added to HP strains liquid medium. At different time points (4,8,12,24,48 hours after culture), 10μl culture medium was removed from each tube, add to 1ml reaction solution, and tested urease activity. After 24 hours,other 10μl culture medium was diluted to different concentrations and then were inoculated on Brucella medium containing calf serum to observe the growth of HP strains and count colony forming units.Results:In solid culture, anti-HP lactobacillus acidophilus supernatant inhibited all HP strains obviousely.In liquid culture, anti-HP lactobacillus acidophilus can inhibit the proliferation in antibiotic- resistant HP strains and urease activity. All three groups results showed that anti-HP lactobacillus acidophilus(LA4 and LA6) have stronger inhibitory effect on HP than standard lactobacillus acidophilus(LA).Conclusion:Anti-HP acidophilus(LA4 and LA6) significantly inhibited antibiotic- resistant HP strains.更多还原