中药对临床型奶牛乳房炎源大肠杆菌的体外抑制及耐药消除研究

发布时间：2019-05-09 19:01

【摘要】：奶牛乳房炎是奶牛乳腺组织受到内、外界环境中的刺激因素如物理、化学、微生物等因素刺激所产生的一种炎性反应,是奶牛生产中的常见病和多发病,而多种病原微生物混合感染是引起奶牛乳房炎发病的主要原因,严重制约了当前奶牛业的发展。奶牛场乳房炎的防控与治疗目前主要依赖抗菌药物,但由于长期和广泛使用抗菌药物,使得细菌耐药性增强现象普遍发生,导致乳汁中抗菌药物残留超标、环境污染严重及乳制品安全受到威胁等问题日益突出,引起了全社会的广泛关注。我国传统中草药具有天然性、多功能性、基本无耐药性、不易在畜禽及其产品中残留、毒副作用小和不污染环境等优点,在奶牛乳房炎防治中的应用越来越受到重视。其中,中草药黄连、黄芩及其复方制剂具有清热解毒、抑菌、抗病毒等作用,在临床上的研究和应用已成为热点。本研究收集陕西省部分地区奶牛场患临床型乳房炎奶牛乳样共95份,采用革兰氏染色镜检、生理生化试验、分子生物学特性检测等方法对其进行大肠杆菌的分离和鉴定;采用二倍稀释法对分离得到的大肠杆菌进行四组中药(黄连、黄芩、黄连黄芩(1:1)水煎剂及双黄连冻干粉针剂)体外抑菌试验;采用扫描电镜及酶标板法检测抑菌效果较好的黄芩和双黄连对细菌胞壁及细菌外观微形态的影响;进一步检测了所分离大肠杆菌对常用14种抗菌药物的耐药情况以及四组中药对大肠杆菌耐药性的消除作用,通过RT-PCR方法对比了四组中药对大肠杆菌所含耐药基因的影响,结果如下:本研究共分离得到大肠杆菌15株。四组中药对所分离15株大肠杆菌均有一定程度的体外抑制作用,药物体外MIC50测定结果显示,双黄连在所选中药中抑菌作用最强,其MIC50为3.90 mg/mL;其次是黄芩,其MIC50为7.81 mg/mL;然后是黄连和黄芩与黄连的组合,其MIC50为15.625 mg/mL。发射场电子扫描显微镜观察大肠杆菌胞体表面微观形态显示随着中药作用时间的延长,大肠杆菌胞体表面完整性破坏越发严重,说明中药黄芩和双黄连均对大肠杆菌有破坏作用;中药培养的大肠杆菌上清液中AKP随着中药作用时间的增加含量为上升趋势,表明中药黄芩和双黄连可增加大肠杆菌细胞壁的通透性,提示黄芩和双黄连主要通过对大肠杆菌细胞壁的破坏及菌体的分解破坏发挥抑制作用。15株大肠杆菌分离株对现阶段临床上普遍使用的大部分抗菌药物均表现出不同程度的耐药,其中对林可霉素、红霉素、青霉素的耐药率高达100%,而对头孢西丁、头孢噻肟的耐药率为0%。经中药培养后各组菌株敏感性普遍增强,其中以黄芩的作用最为显著;对单种抗菌药物(如万古霉素)耐药率降低最多的是黄芩(53%),其次是双黄连(40%)。15株大肠杆菌检测出β-内酰胺酶(Extended spectrumβ-lactamase,ESBL)基因型两种blaTEM和blaSHV,检出率分别为40%和13.3%;检测出质粒介导的喹诺酮耐药(Plasmid-mediated quinolone resistance,PMQR)基因型三种qnrB,qnrD和qnrS检出率均为13.3%。经中药作用后,耐药基因均呈现不同的减弱或消除,以黄芩对5种耐药基因的消除效果最好,黄芩黄连(1:1)次之。以上试验结果表明,黄连、黄芩、双黄连对临床型奶牛乳房炎乳源大肠杆菌具有一定的体外抑菌作用,黄芩、双黄连主要通过破坏大肠杆菌细胞壁完整性及分解破坏菌体发挥抑菌作用。药敏试验和分子生物学检测表明,中药黄连、黄芩、双黄连对大肠杆菌的耐药性和耐药基因均有不同程度的减弱或消除作用。
[Abstract]:The mastitis of the milk cow is an inflammatory reaction caused by the stimulation factors such as physical, chemical and micro-organisms in the dairy cow's breast tissue, which is a common disease and a disease in the production of the dairy cow, The mixed infection of a plurality of pathogenic microorganisms is the main cause of the occurrence of the mastitis of the dairy cow, and the development of the current dairy cattle industry is seriously restricted. The prevention and control and treatment of the mastitis of the dairy cattle field are mainly dependent on the antibacterial drugs, The problem of serious environmental pollution and the threat of the safety of dairy products has become more and more prominent, which has attracted wide attention from the whole society. The traditional Chinese herbal medicine has the advantages of natural nature, multi-function, basically no drug resistance, no residue in the livestock and poultry and the products thereof, small toxic and side effect, no environmental pollution and the like, and the application of the traditional Chinese herbal medicine in the prevention and treatment of the mastitis of the dairy cow is more and more important. The Chinese herbal medicine coptis, the Chinese medicinal preparation and the compound preparation have the effects of clearing away heat and toxic materials, inhibiting bacteria and resisting virus, and has become a hot spot in clinical research and application. In this study,95 samples of dairy cows with clinical mastitis were collected in the dairy cattle farm in some parts of Shaanxi, and the isolation and identification of E. coli were carried out by the methods of Gram staining, physiological and biochemical test and molecular biological characteristic test. in vitro antibacterial test of four groups of traditional Chinese medicine (coptis root, yellow root, coptis chinensis (1:1), and Shuanghuanglian freeze-dried powder injection) are carried out by adopting a double dilution method; The effects of yellow and double coptis on the micromorphology of the bacterial cell wall and the bacterial appearance are detected by using the scanning electron microscope and the enzyme labeling method, the drug resistance of the isolated E. coli to the common 14 antibacterial drugs and the elimination effect of the four groups of the traditional Chinese medicine on the drug resistance of the Escherichia coli are further detected, The effect of four groups of traditional Chinese medicine on the resistance gene contained in E. coli was compared by RT-PCR. The results were as follows:15 strains of E. coli were isolated. The four groups of traditional Chinese medicines have a certain degree of in-vitro inhibition on the isolated 15 strains of E. coli. The results of the determination of the in vitro MIC50 of the drug show that the inhibitory effect of the Shuanghuanglian in the selected Chinese medicine is the strongest, the MIC50 of the two coptis is 3.90 mg/ mL, the second is the yellow root, the MIC50 of the traditional Chinese medicine is 7.81 mg/ mL, and then is a combination of the coptis and the Chinese goldthread and the Chinese goldthread. Its MIC50 is 15.625 mg/ mL. The micromorphology of the surface of E. coli cell was observed by the electron-scanning microscope of the launch site, and the surface integrity of the E. coli cell was more and more serious with the extension of the time of the traditional Chinese medicine. The content of AKP in the supernatant of E. coli cultured by traditional Chinese medicine is the rising trend with the increasing of the action time of the traditional Chinese medicine, indicating that the traditional Chinese medicine yellow and double coptis can increase the permeability of the cell wall of the Escherichia coli, The results showed that Huang-Huang and Shuanghuanglian had an inhibitory effect on the destruction of the cell wall of E. coli and the decomposition and destruction of the cells. The 15 strains of E. coli isolates showed different resistance to most of the antibacterial drugs commonly used at the present stage, including lincomycin and erythromycin. The resistance rate of the penicillin is as high as 100%, and the resistance rate to cefoxitin and cefixime is 0%. the sensitivity of each group of strains after the culture of the traditional Chinese medicine is generally enhanced, and the effect of the yellow-root is the most significant; the drug resistance rate of a single antibacterial agent (such as vancomycin) is reduced to a maximum of yellow-root (53%), followed by double coptis (40%), and 15 strains of Escherichia coli are used for detecting an extended spectrum-lactamase, The detection rate was 40% and 13.3%, respectively. The detection rate of three qnrB, qnrD and qnrS was 13.3%. After the action of the traditional Chinese medicine, the drug-resistant gene has different attenuation or elimination, and the effect of the yellow rice on the five drug-resistant genes is the best, and the Huanglian Coptis (1:1) is the second. The results of the above test show that Coptis chinensis, Huang-Huang and Shuanghuanglian have a certain in-vitro antibacterial effect on the clinical-type dairy cow mastitis milk-source E. coli. The drug sensitivity test and molecular biology test show that the drug resistance and the drug resistance gene of the Chinese medicine coptis root, the yellow root and the Shuanghuanglian have different degrees of attenuation or elimination.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S858.23

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