【摘要】：本研究采用傳統(tǒng)微生物分離方法，分別以9種抗生素為唯一碳源，從健康人糞便樣品中，分離腸道抗生素同化菌。根據(jù)目的菌群在兒童、青年、老年三類(lèi)健康人群中的檢出結(jié)果，統(tǒng)計(jì)分析腸道抗生素同化菌的存在隨宿主年齡變化的趨勢(shì)。使用微量培養(yǎng)法，測(cè)定抗生素同化菌對(duì)不同濃度梯度抗生素的同化能力，并測(cè)試其對(duì)9種不同抗生素的同化譜。采用K-B紙片擴(kuò)散法測(cè)試目的菌株對(duì)14種抗生素的耐藥譜。本文為微生物的抗生素耐藥機(jī)制及抗生素藥物在腸道環(huán)境的轉(zhuǎn)歸研究提供了實(shí)驗(yàn)基礎(chǔ)。 應(yīng)用16S rDNA分子鑒定方法，對(duì)分離出的目的菌株進(jìn)行菌種鑒定，分析目的菌株的系統(tǒng)發(fā)育關(guān)系，及不同年齡人群中目的菌株的種屬分布特點(diǎn)。16S rDNA分子鑒定結(jié)果顯示，分離到的115株同化菌株分布于變形菌門(mén)和厚壁菌門(mén)。主要分屬于3個(gè)菌屬：克雷伯氏屬、埃希氏菌屬、腸球菌屬，少數(shù)菌株分屬于其它7個(gè)菌屬：泛生菌屬、志賀菌屬、沙雷氏菌屬、黃單胞菌屬、假單胞菌屬、伯克氏菌屬和芽孢桿菌屬。多數(shù)菌株為腸道正常菌群，少數(shù)為條件致病菌，沒(méi)有發(fā)現(xiàn)致病菌存在。 針對(duì)5株氯霉素同化菌株，對(duì)菌株形態(tài)、生理生化特征進(jìn)行分析。分離到的5株氯霉素同化菌株均為革蘭氏陰性菌。其中，菌株編號(hào)為I-11-CHL-1，I-7-CHL-1，II-2-CHL-3，II-3-CHL-1的4株氯霉素同化菌屬于克雷伯氏屬，菌株I-10-CHL屬于大腸埃希氏屬。在氯霉素含量為20mg/L的唯一碳源培養(yǎng)基中，培養(yǎng)13天后，5株同化菌株分別不同程度的降解氯霉素，其中降解率最高為45.5%，平均降解率為16.6%。5株氯霉素同化菌株對(duì)氯霉素表現(xiàn)敏感。 本實(shí)驗(yàn)對(duì)菌株I-10-CHL在單一環(huán)境因素，包括氯霉素初始含量、pH值及外加碳源等影響下，其生長(zhǎng)速率和氯霉素降解率的變化規(guī)律進(jìn)行了詳細(xì)研究。氯霉素初始濃度越高，菌株I-10-CHL對(duì)氯霉素的降解率越低；在pH=7.0的環(huán)境下生長(zhǎng)較好，，菌密度最大，氯霉素降解率也最高；按0.1‰的添加比，分別添加5種第二碳源：苯甲酸鈉、乙酰胺、葡萄糖、三氯乙酸、水楊酸，發(fā)現(xiàn)添加葡萄糖、乙酰胺時(shí)，可提高氯霉素的生物降解率。 利用UV-Vis及LC/MS對(duì)菌株I-10-CHL降解氯霉素的中間產(chǎn)物進(jìn)行分析研究，初步推斷了氯霉素的微生物降解途徑。氯霉素可能的降解途徑為：氯霉素經(jīng)酰胺酶水解生成中間產(chǎn)物1-硝基苯-2-氨基-1,3-丙二醇及二氯乙酸，再經(jīng)歷復(fù)雜的中間反應(yīng)后，最后轉(zhuǎn)化為二氧化碳、水、銨離子和氯離子。
[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

本文編號(hào)：2368084