潜在益生乳杆菌的抗生素敏感性研究

发布时间：2018-06-18 03:38

本文选题：乳杆菌 + 耐药性　；参考：《河北农业大学》2012年硕士论文

【摘要】：乳杆菌在自然界分布广泛,且作为益生菌广泛应用于食品、饲料和医药卫生等相关领域。近年来,越来越多的研究表明,抗生素的广泛应用在治疗疾病的同时,也使得环境中耐药菌株的种类及数目增加,携带有可转移耐药因子的益生菌应用于生产,将严重影响到食品的安全性。本文对从食品环境中分离保存的34株乳杆菌的抗生素敏感性进行了研究,首先从具有较高耐药性菌株中检测是否存在质粒DNA,探讨了在胃肠条件下含质粒菌株生长的耐受性;采用质粒消除法分析了菌株质粒谱与耐药谱之间的联系,探索了乳杆菌耐抗生素的分子机制,旨在为益生乳杆菌安全性评价体系建立提供理论依据。主要结果如下: 1、抗生素敏感性测定结果表明,供试乳杆菌菌株对杆菌肽、多粘菌素B、卡那霉素以及萘啶酸的耐药率达到了100%;对万古霉素的耐药率为82.4%(28/34);对链霉素的耐药率为91.2%(31/34);对庆大霉素的耐药率为88.2%(30/34),表明供试菌株对这几种抗生素具有普遍的耐药性,耐药谱的分布表现出种间差异性。供试菌株对青霉素的耐药率为38.2%(13/34);对头孢噻吩的耐药率为17.6%(6/34);对氨苄西林的耐药率为41.2%(14/34);对四环素和氯霉素的耐药率为8.8%(3/34);对利福平的耐药率是29.4%(10/34),表明供试菌株对这几种抗生素相对比较敏感,且敏感性分布具有种属特异性。 2、选择耐药性相对较强的16株菌提取质粒,最终确定有6株含有质粒,其中菌株CH8、Lp-A和Lp-4为戊糖乳杆菌,LLB为瑞士乳杆菌,LL为嗜酸乳杆菌,L11为植物乳杆菌。模拟胃肠环境生存实验表明,含质粒菌株CH8、Lp-A、LLB、LL和L11能够在胃肠环境下生存下来,这意味着质粒上携带抗性基因的菌株或许会对寄主健康带来潜在危害。 3、利用质粒消除法证实菌株CH8质粒上可能含有氯霉素和β-内酰胺类的抗性基因。PCR分析证实菌株CH8上拥有大小为10kb左右的两条质粒,测序表明两条质粒上含有β-内酰胺类的抗性基因blr。进一步地,将来自菌株CH8的耐药质粒转化于对氯霉素敏感的大肠杆菌HB101中,观察到大肠杆菌HB101在含有氯霉素的培养基上能够生长。综上,在敏感性及耐药性质粒检测基础上,评价含耐药质粒菌株在胃肠环境的生存能力,将在肠胃逆境下生存菌株进行质粒消除,以此探讨质粒谱与耐药谱之间的联系,再将菌株质粒转化到抗生素敏感的大肠杆菌细胞中,可以形成一条新的潜在益生乳杆菌抗生素敏感性的安全性评价程序。
[Abstract]:Lactobacillus is widely distributed in nature and is widely used as probiotics in food, feed, medicine and hygiene. In recent years, more and more studies have shown that the widespread use of antibiotics in the treatment of diseases, but also to increase the variety and number of drug-resistant strains in the environment, carrying transferable resistance factors of probiotics used in production, Will seriously affect the safety of food. In this paper, the antibiotic sensitivity of 34 strains of Lactobacillus isolated from food environment was studied. The relationship between plasmid profile and drug resistance spectrum of Lactobacillus was analyzed by plasmid elimination method, and the molecular mechanism of antibiotic resistance of Lactobacillus was explored in order to provide theoretical basis for the establishment of safety evaluation system of probiotic Lactobacillus. The main results are as follows: 1. The results of antibiotic sensitivity test showed that Lactobacillus strains were resistant to bacilli peptide. The resistance rates of polymyxin B, kanamycin and naphthalic acid reached 100; vancomycin resistance rate was 82.4- 28 / 34%; streptomycin resistance rate was 91.21% / 34%; and gentamicin resistance rate was 88.2% 30% 34%, indicating that the strains tested had universal resistance to these antibiotics. The distribution of drug resistance spectrum showed interspecific differences. The resistance rate of the tested strains to penicillin was 38.2% / 34%; to cefthiophene was 17.6 / 634%; to ampicillin was 41.22% / 34%; to tetracycline and chloramphenicol was 8.88% / 34%; and to rifampicin was 29.44% to rifampicin, indicating that the strains were resistant to these strains. Antibiotics are relatively sensitive. The sensitivity distribution was species-specific. 2. 16 strains with relatively strong drug resistance were selected to extract plasmids, and 6 strains were found to contain plasmids. Among them, strains CH8 Lp-A and Lp-4 were Lactobacillus pentanaccharis, LLB, Lactobacillus Switzerland, LLL, Lactobacillus acidophilus, Lactobacillus acidophilus, L11, Lactobacillus plantarum. The experimental results of simulated gastrointestinal survival showed that plasmid strains CH8G Lp-An LLBNLL and L11 could survive in gastrointestinal environment. This means that the strain carrying the resistance gene on the plasmid may be a potential hazard to the host health. 3. The plasmid elimination method was used to confirm that the plasmid of strain CH8 might contain chloramphenicol and 尾-lactam resistance genes. PCR It was confirmed that the strain CH8 had two plasmids about the size of 10kb. Sequencing showed that the two plasmids contained 尾 -lactam resistance gene blr. Furthermore, the resistant plasmid from strain CH8 was transformed into Escherichia coli HB101, which was sensitive to chloramphenicol. It was observed that E. coli HB101 could grow on the medium containing chloramphenicol. On the basis of sensitivity and drug resistance plasmid detection, the survivability of drug-resistant plasmid strains in gastrointestinal environment was evaluated, and the plasmid elimination was carried out under gastrointestinal stress, so as to explore the relationship between plasmid profile and drug resistance spectrum. A new procedure for evaluating the antibiotic sensitivity of Lactobacillus probiotics was established by transforming the plasmid into E. coli cells sensitive to antibiotics.
【学位授予单位】：河北农业大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R378

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