人体肠道中抗生素同化菌特性的研究

发布时间：2018-12-08 09:05

【摘要】：本研究采用传统微生物分离方法，分别以9种抗生素为唯一碳源，从健康人粪便样品中，分离肠道抗生素同化菌。根据目的菌群在儿童、青年、老年三类健康人群中的检出结果，统计分析肠道抗生素同化菌的存在随宿主年龄变化的趋势。使用微量培养法，测定抗生素同化菌对不同浓度梯度抗生素的同化能力，并测试其对9种不同抗生素的同化谱。采用K-B纸片扩散法测试目的菌株对14种抗生素的耐药谱。本文为微生物的抗生素耐药机制及抗生素药物在肠道环境的转归研究提供了实验基础。 应用16S rDNA分子鉴定方法，对分离出的目的菌株进行菌种鉴定，分析目的菌株的系统发育关系，及不同年龄人群中目的菌株的种属分布特点。16S rDNA分子鉴定结果显示，分离到的115株同化菌株分布于变形菌门和厚壁菌门。主要分属于3个菌属：克雷伯氏属、埃希氏菌属、肠球菌属，少数菌株分属于其它7个菌属：泛生菌属、志贺菌属、沙雷氏菌属、黄单胞菌属、假单胞菌属、伯克氏菌属和芽孢杆菌属。多数菌株为肠道正常菌群，少数为条件致病菌，没有发现致病菌存在。 针对5株氯霉素同化菌株，对菌株形态、生理生化特征进行分析。分离到的5株氯霉素同化菌株均为革兰氏阴性菌。其中，菌株编号为I-11-CHL-1，I-7-CHL-1，II-2-CHL-3，II-3-CHL-1的4株氯霉素同化菌属于克雷伯氏属，菌株I-10-CHL属于大肠埃希氏属。在氯霉素含量为20mg/L的唯一碳源培养基中，培养13天后，5株同化菌株分别不同程度的降解氯霉素，其中降解率最高为45.5%，平均降解率为16.6%。5株氯霉素同化菌株对氯霉素表现敏感。 本实验对菌株I-10-CHL在单一环境因素，包括氯霉素初始含量、pH值及外加碳源等影响下，其生长速率和氯霉素降解率的变化规律进行了详细研究。氯霉素初始浓度越高，菌株I-10-CHL对氯霉素的降解率越低；在pH=7.0的环境下生长较好，，菌密度最大，氯霉素降解率也最高；按0.1‰的添加比，分别添加5种第二碳源：苯甲酸钠、乙酰胺、葡萄糖、三氯乙酸、水杨酸，发现添加葡萄糖、乙酰胺时，可提高氯霉素的生物降解率。 利用UV-Vis及LC/MS对菌株I-10-CHL降解氯霉素的中间产物进行分析研究，初步推断了氯霉素的微生物降解途径。氯霉素可能的降解途径为：氯霉素经酰胺酶水解生成中间产物1-硝基苯-2-氨基-1,3-丙二醇及二氯乙酸，再经历复杂的中间反应后，最后转化为二氧化碳、水、铵离子和氯离子。
[Abstract]:In this study, 9 antibiotics were used as the sole carbon source to isolate enteric antibiotic assimilating bacteria from fecal samples of healthy people. According to the results of the detection of bacterial flora in children, young people and old people, the tendency of antibiotic assimilation bacteria in intestinal tract with host age was analyzed statistically. The assimilation ability of antibiotic assimilation bacteria to different concentration gradient antibiotics was measured by microculture method, and the assimilation spectrum of 9 different antibiotics was tested. The resistance spectrum of the target strain to 14 antibiotics was tested by K-B disk diffusion method. This study provides an experimental basis for the mechanism of antibiotic resistance of microorganisms and the outcome of antibiotics in intestinal environment. The 16s rDNA molecular identification method was used to identify the isolated target strains, the phylogenetic relationship of the target strains and the distribution characteristics of the target strains in different age groups were analyzed. The results of 16s rDNA molecular identification showed that, 115 assimilative strains were isolated and distributed in the phylum of Proteus and phylum thunbergiformis. It mainly belongs to three genera: Klebsiella, Escherichia, Enterococcus, and a few strains belong to the other 7 genera: Phagophyte, Shigella, Shareh, Xanthomonas, Pseudomonas. Burke's and Bacillus. Most of the strains were normal intestinal flora, a few were conditional pathogens, and no pathogenic bacteria were found. The morphological, physiological and biochemical characteristics of 5 chloramphenicol assimilated strains were analyzed. The 5 chloramphenicol assimilated strains were all Gram-negative bacteria. Among them, four strains of chloramphenicol, which were identified as I-11-CHL-1, I-7-CHL-1, II-2-CHL-3, II-3-CHL-1, belong to Klebsiella and I-10-CHL belong to Escherichia coli. After 13 days of culture, 5 assimilated strains degraded chloramphenicol in different degrees, and the highest degradation rate of chloramphenicol was 45.5% in the medium with chloramphenicol content being the only carbon source of 20mg/L. The average degradation rate was 16.6.5 strain of chloramphenicol assimilated strain was sensitive to chloramphenicol. The growth rate and chloramphenicol degradation rate of strain I-10-CHL were studied in detail under the influence of a single environmental factor, including the initial content of chloramphenicol, the value of pH and the addition of carbon source. The higher the initial concentration of chloramphenicol, the lower the degradation rate of chloramphenicol by strain I-10-CHL. According to the ratio of 0.1 鈥

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