致病性E.coli分离鉴定及其蒙药印菌成分筛选和作用的研究

发布时间：2018-03-30 12:04

本文选题：犊牛腹泻　切入点：大肠杆菌　出处：《内蒙古农业大学》2015年硕士论文

【摘要】：长期使用抗生素会引发许多如畜产品内药物残留、耐药性等现象。蒙药是一种纯天然的药物,在课题组前期研究中发现,蒙药巴布-7抑菌作用强,疗效显著。为了筛选出该复方诸多成分中,有效抑菌成分以及最佳体内外抑菌组方,并揭示其作用机理,为此进行了本研究。试验结果如下：1.在调查呼和浩特市周边致病性大肠杆菌的血清学分布时,从呼和浩特市周边某牛场采集了117份粪样,进行分离培养后得到97株疑似大肠杆菌,进行形态学观察与生化鉴定,确定其中65株为大肠杆菌。进行“O”血清型鉴定,分离出0145、O152、0167三种血清型,分别占65株大肠杆菌的1.96%、1.96%、7.84%。将这65株大肠杆菌进行PCR鉴定后发现又多了新的血清型026和083,这两种血清型在血清型试验中没有检测到,该结果说明了PCR方法的敏感性。将这65株大肠杆菌进行动物致病性试验,其中有51株为强致病性大肠杆菌,占采样量的40%。说明强致病性大肠杆菌分布较广。2.采用牛津杯法,初步筛选出蒙药巴布-7的6种抑菌有效成分中的5种对致犊牛腹泻病大肠杆菌均有抑菌作用,测定最低抑菌浓度(MIC)与分级抑菌浓度(FIC)指数,确定5种药物互相都无拮抗作用,适宜配伍。5种药物对大肠杆菌的MIC分别是茜草素为5mg/mL,大黄酸为20mg/mL,大黄素为20mg/mL,槲皮素为7.5mg/mL,没食子酸为7.5mg/mL3.首先得出对大肠杆菌具有最佳抑菌作用的药物,针对大肠杆菌5种药物均用药。之后确定各药物之间的剂量配比关系。得出对大肠杆菌抑菌效果最好的药物组合为A2B1C3D2E2.4.采用微量稀释法测定抑菌组方的MIC,得出蒙药抑菌组方对大肠杆菌的最低抑菌浓度为0.675mg/mL,通过测定小鼠体内保护率得出该抑菌组方保护率为50%,说明蒙药抑菌成分组方体内抑菌保护率与阳性对照组环丙沙星66.7%接近。5.为了给优化后的抑菌组方的抑菌作用提供理论依据,试验通过邻硝基苯β-D-半乳吡喃糖苷(ONPG)法测定其对E. coli细胞膜渗透性的影响,并测定其对E. coli电导率、细胞壁的影响,进一步研究了抑菌组方对大肠杆菌细胞膜的影响。结果表明,大肠杆菌经过抑菌组方作用后,细胞膜的通透性、溶液的电导率与碱性磷酸酶(AKP)的含量均增加。证明药物可能是通过破坏细菌细胞膜导致其渗透性增加,从而发挥其抑菌作用。6.为了测定蒙药抑菌成分复方的抗菌谱,通过体外抑菌直径的测定与最低抑菌浓度的测定,得出该抑菌组方对大肠杆菌、金黄色葡萄球菌等细菌均有抑制作用。
[Abstract]:Long-term use of antibiotics can lead to many phenomena such as drug residues in animal products, drug resistance, etc. Mongolian medicine is a pure natural drug, in the previous research group found that the Mongolian drug Babu-7 strong bacteriostatic effect, In order to screen out the effective antimicrobial components and the best in vitro and in vivo antimicrobial components of the compound, and to reveal the mechanism of its action, The results are as follows: 1.When investigating the serological distribution of pathogenic Escherichia coli around Hohhot, 117 feces were collected from a cattle farm around Hohhot. After isolation and culture, 97 strains of suspected Escherichia coli were obtained, and 65 of them were identified as E. coli by morphological observation and biochemical identification. The new serotypes 026 and 083 were found after the PCR identification of the 65 strains of Escherichia coli. These two serotypes were not detected in the serotype test. The results showed the sensitivity of the PCR method. Among the 65 strains of Escherichia coli, 51 were strong pathogenic Escherichia coli, accounting for 40% of the sample size. 2. 2. Oxford Cup method was used to test the pathogenicity of Escherichia coli. Five of the six antimicrobial active components of Mongolian drug Babu-7 had bacteriostatic effect on Escherichia coli caused by diarrhea disease in calves. The index of minimal inhibitory concentration (MIC) and fractionated inhibitory concentration (FICI) were determined, and the five drugs had no antagonistic effect on each other. The suitable MIC for E. coli was 5 mg / mL for alizarin, 20 mg / mL for Rhein, 20 mg / mL for emodin, 7.5 mg / mL for quercetin and 7.5 mg / mL for Gallic acid, respectively. The dose-ratio relationship between each drug was determined. The best drug combination for Escherichia coli was A _ 2B _ 1C _ 3D _ 2E _ 2.4.The microdilution method was used to determine the antimicrobial composition and the Mongolian medicine was obtained. The minimum inhibitory concentration of bacteriostasis prescription against Escherichia coli was 0.675 mg / mL. The protective rate of the antimicrobial component was 50 by measuring the protective rate in vivo of mice, indicating that the bacteriostatic protection rate in Mongolian medicine was 66.7% compared with ciprofloxacin in positive control group. In order to provide a theoretical basis for the bacteriostatic effect of the optimized antimicrobial prescription, The effects of o-nitrobenzene 尾 -Dgalactopyranoside on the permeability of E. coli cell membrane, the conductivity of E. coli and the cell wall were measured by the method of o-nitrobenzene 尾 -D- galactopyranoside. The effect of antimicrobial prescription on the membrane of Escherichia coli was further studied. Both the electrical conductivity of the solution and the content of alkaline phosphatase (AKP) increased. It was proved that the drug may cause the increase of its permeability by destroying the bacterial cell membrane, thus exerting its bacteriostatic effect .6. in order to determine the antibacterial spectrum of the compound of the bacteriostasis component of Mongolian medicine, Through the determination of the antibacterial diameter in vitro and the determination of the minimum inhibitory concentration, it was concluded that the antimicrobial compound had inhibitory effect on Escherichia coli, Staphylococcus aureus and other bacteria.
【学位授予单位】：内蒙古农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.61;S853.7

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