种鸡场大肠杆菌噬菌体的分析与临床应用

发布时间：2018-05-05 22:04

本文选题：大肠杆菌 + MLST分型　；参考：《山东大学》2017年硕士论文

【摘要】：遏制细菌耐药性行动计划(2016-2020)是全民的共识与国家的意志的体现,需要实施切实的技术措施。除了依据可靠的药敏试验结果精准用药外,噬菌体以其特异性强、与抗生素无交叉抗药性、无残留、对人畜无感染性、容易筛选获得等独特优势成为潜力巨大的选项。本文从山东省种鸡场、商品鸡场采集病料和粪样筛选大肠杆菌的流行菌株,完善药敏试验方法,预警抗药性菌株;筛选裂解性噬菌体,分析其生物学性质和基因结构,通过注射、口服方式进行治疗试验,以期控制种鸡场致病性大肠杆菌的感染和流行。使用麦康凯培养基从临床样品和粪样中筛选出疑似大肠杆菌,经大肠杆菌特异性引物phoA共鉴定出大肠杆菌株117株。多位点序列分型(multilocus sequence typing,MLST)结果表明共有 65 个 ST 型,其中 ST155、ST156 与 ST69居多,另发现两个新ST型。线性梯度平板能精确地测定MIC,推算药物的原始浓度,能更好地保证质控试验的可靠性。当按照美国临床实验室标准委员会(Clinical Laboratory Standards Institute,CLSI)或欧盟药敏试验委员会(European Committee on Antimicrobial Susceptibility Testing,EUCAST)的 S(Susceptible)、I(Intermediate)、R(Resistant)范围标准设置0-S、S-R、R-8R这样三个线性梯度,首先可以判断菌株敏感性范围及细菌群落精确的MIC值,并能分离出抗性菌株,预警该抗生素使用后可能出现的抗药性,进而指导临床药物的选择和应用。如果自动化生产即用型梯度平板,将便利临床药敏试验,改进抗药性筛选技术。以半固体双层平板噬菌斑法针对上述流行性耐药大肠杆菌共分离纯化到大肠杆菌噬菌体143株。挑选主要ST型菌株ST155对应的3株大肠杆菌噬菌体进行了测序和电镜观察,噬菌体均属于有尾目、肌尾科T4噬菌体类型,由二十面体头部与尾部组成,其中vB-EcolP-YSFl为新型噬菌体,与已测序噬菌体只有11%的相似性。vB-EcolP-XT4、vB-EcolP-RZⅢ与vB-EcolP-YSF1生物学特性分析表明,噬菌体效价均可达到10~9pfu/ml,且热稳定性较好,在60℃温度下保温1 h后噬菌体效价仍可达到10~7pfu/ml以上;最适生长温度均在37 ℃左右;在pH值为4-10的区间内能保持较好的裂解活性,最适pH偏中性;噬菌体的最佳感染复数一般在0.1-1之间,噬菌体在感染宿主菌后的潜伏期一般为20 min。通过大肠杆菌含有的毒力基因数目与其半数致死量确定攻毒菌株,以棋盘法试验优化最佳感染复数,确定大肠杆菌噬菌体vB-EcolP-YSF1与大肠杆菌E.YSF-1肝作用的最佳浓度比,噬菌体治疗采用胸肌注射肉鸡,表现出了较好的治疗效果,并随着噬菌体效价的提高,肉鸡的死亡率降低;噬菌体口服治疗SPF鸡实验采用同时口服噬菌体vB-EcolP-RZⅢ与高抗的致病性大肠杆菌,噬菌体治疗组比只口服致病菌组粪样中该高抗的致病性大肠杆菌降低了至少1个数量级,这在实际生产条件下具有良好的临床应用价值,为后期噬菌体治疗禽源大肠杆菌病奠定基础。未来将根据上述试验结果在种鸡场进行大肠杆菌大规模的环境净化与大肠杆菌病防治试验。
[Abstract]:The action plan to curb bacterial resistance (2016-2020) is the consensus of the whole people and the will of the country. It is necessary to implement practical technical measures. In addition to the accurate drug use according to the results of reliable drug sensitivity test, the bacteriophage is strong, no cross resistance to antibiotics, no residue, no infection to human and animal, and easy selection and so on. Advantage has become an option with great potential. In this paper, the epidemic strains of Escherichia coli were collected from the chicken farm of Shandong Province, the poultry farms were collected from the poultry farms and the feces samples were collected, the drug sensitivity test method was perfected, the resistant strains were early warning, the lysate phage was screened, the biological properties and the gene structure were analyzed, the treatment test was carried out by injection and oral administration, in order to control it. The infection and epidemic of pathogenic Escherichia coli in the chicken farm. The Escherichia coli was screened from clinical samples and feces by Makanke medium, and 117 Escherichia coli strains were identified by phoA specific primers. The results of multilocus sequence typing (MLST) showed that there were 65 ST types, including ST155, ST1. 56 and ST69 are many, and two new ST types are found. The linear gradient plate can accurately measure the MIC and calculate the original concentration of the drug. It can better guarantee the reliability of the quality control test. Timicrobial Susceptibility Testing, EUCAST) S (Susceptible), I (Intermediate), R (Resistant) range standard set 0-S, S-R, and such three linear gradients, first can determine the strain sensitivity range and the bacterial community accurate values, and can separate the resistant strain, early warning of the antibiotic may appear after the use of resistance, into the drug. To guide the selection and application of clinical drugs. If automated production is used as a gradient plate, the clinical drug sensitivity test will be facilitated and the resistance screening technology is improved. 143 strains of Escherichia coli are isolated and purified to the above epidemic resistant Escherichia coli by semi solid bilayer plate plaque method, and 3 of the main ST strains are selected for the selection of 3 of the main strains of Escherichia coli. The bacteriophages of Escherichia coli were sequenced and observed by electron microscope. The phage belonged to the phage of the caudate. The type of T4 phage was made up of the head and tail of the twenty facet body. The vB-EcolP-YSFl was a new phage, and only 11% of the phage had been sequenced.VB-EcolP-XT4, and the biological characteristics of vB-EcolP-RZ III and vB-EcolP-YSF1 were analyzed. The bacteriophage titer can reach 10~9pfu/ml, and the thermal stability is better. The phage titer can still reach more than 10~7pfu/ml after 1 h heat preservation at 60 C. The optimum growth temperature is about 37. The optimum cracking activity in the interval of pH value is 4-10, and the optimum pH is neutral. The best number of phage infection is generally between 0.1-1, The latent period of phage infection after infection is usually 20 min. through the number of virulence genes and half lethal dose of Escherichia coli to determine the virulence strain. The optimal number of infection is optimized by chessboard test, and the optimal concentration ratio of Escherichia coli vB-EcolP-YSF1 and Escherichia coli E.YSF-1 liver is determined, and phage therapy is used. The breast muscle injection of broilers showed a better therapeutic effect, and with the increase of phage titer, the mortality of broilers decreased, and phage SPF chickens were treated with oral phage vB-EcolP-RZ III and highly resistant pathogenic Escherichia coli, and the phage therapy group was more resistant to the pathogenic Escherichia coli in the fecal samples of the oral pathogenic bacteria group. The bacilli decreased at least 1 orders of magnitude, which was of good clinical value under actual production conditions and laid the foundation for the later phage treatment of avian Escherichia coli. In the future, large scale environmental purification and colibacillosis prevention and control test will be carried out in the chicken farm based on the experimental results.

【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.6

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