一株牛源肺炎克雷伯菌的分离鉴定及其基因组初步分析

发布时间：2018-05-12 16:58

本文选题：肺炎克雷伯菌 + 分离　；参考：《甘肃农业大学》2017年硕士论文

【摘要】：肺炎克雷伯菌(Klebsiella pneumoniae,K.pneumoniae)是肠杆菌科克雷伯菌属的一种常见条件性致病菌,也是一种重要的人兽共患病原菌,其感染仅次于大肠杆菌和绿脓杆菌。目前,该菌呈世界性分布,主要存在于人和动物的肠道、呼吸道和泌尿生殖道及水、土壤和谷物中,当机体免疫力低下或长期大量使用抗菌素导致菌群失调时可引起肺炎、肠炎、脑膜炎、腹膜炎、眼炎以及泌尿生殖道炎症等多种疾病等,严重的甚至会引发败血症。近年来,由于抗生素在临床上长期、广泛的使用,使得肺炎克雷伯菌的耐药菌株不断增多,给临床抗感染治疗带来了严峻的挑战。因此,开展肺炎克雷伯菌相关的研究,将为该菌感染相关疾病防治新方法及新措施的研究和开发奠定基础。本研究以发生新生犊牛腹泻和呼吸道症状为主疫情的甘肃省兰州市榆中县某牛场病死犊牛内脏组织为病料,通过病原的分离及鉴定,确定该次疫情是由肺炎克雷伯菌感染所致。基于此,对分离菌株生物学特性进行了研究,最后对其基因组进行了生物信息学的初步分析,从而为肺炎克雷伯菌致病机制及免疫机理的深入研究奠定了一定基础。研究内容及结果主要包括如下几个方面。1.肺炎克雷伯菌YZ株的分离及鉴定基于临床综合诊断,从送检病料中进行了病原菌的分离,并在纯化的基础上完成了致病性试验、药敏试验、生化试验、16S rRNA基因、khe基因及部分毒力基因的扩增及序列分析。结果表明,除H2S试验外,分离菌其他生化特征均符合肺炎克雷伯菌,且其16S rRNA基因和khe基因序列与所有已知序列的肺炎克雷伯菌的同源性均高达99%,而该菌三种不同毒力基因的序列与肺炎克雷伯菌其他菌株的同源性亦高达95%以上。基于此,确定分离菌为肺炎克雷伯菌,且将其定名为YZ株。2.肺炎克雷伯菌YZ株的生物学特性研究在分离及鉴定的基础上,完成了该菌荚膜染色特性,生长曲线测定,生物被膜形成的检测,半数致死量的测定及免疫保护性试验等的研究。结果显示:分离菌不能有效形成荚膜;从绘制的生长曲线可以看出该分离菌在0~4 h处于迟缓期,4 h后进入对数期生长期,10~18 h为稳定期,18 h以后进入衰亡期;生物被膜的定性和定量测定结果显示该分离菌能够形成一定的生物膜;经寇氏改良法计算,得知该菌的半数致死量为1.38×108 CFU,毒力相对较弱;分离菌培养物灭活后按不同浓度(1×107 cfu/mL~1×1010 cfu/mL)配制疫苗,接种小白鼠后未出现异常现象,说明所配疫苗安全性良好;攻毒试验表明当配苗浓度为1×1010 cfu/mL时,保护率即可达到100%,表明分离菌具有良好的免疫保护作用,且免疫后小鼠抗血清ELISA效价高达1:3 200以上。3.肺炎克雷伯菌YZ株的基因组分析为了解分离菌荚膜缺陷的原因及其致病机制,本研究完成了其基因组框架图并对测序结果进行了初步分析。结果显示,分离菌基因组长度为5 336 251 bp,GC含量为57.444%,含有120个Scaffold,166个Contig,共预测到80个tRNA,3个rRNA,基因组中共含有5 205个基因,通过与该属其他菌株比对,发现该菌中与荚膜多糖的转移和表面装配有关的四个保守基因中缺少wzb基因与wzc基因。另外,在荚膜合成中起不同作用的调控子中,仅发现负调控作用基因Fur、crp和SugE的存在。而毒力基因中,未发现rmpA、magA、wabG等基因,且在众多预测基因中还存在一些功能未知的基因。上述结果为进一步研究分离菌荚膜形成缺陷的原因及阐明其致病机制奠定了基础。
[Abstract]:Klebsiella pneumoniae (Klebsiella pneumoniae, K.pneumoniae) is a common conditional pathogenic bacteria of the genus Enterobacteriaceae. It is also an important zoonosis. The infection is second only to Escherichia coli and Pseudomonas aeruginosa. At present, the bacterium is distributed worldwide, mainly in the intestines, respiratory and urinary tract of human and animal. There are many diseases such as pneumonia, enteritis, meningitis, peritonitis, ophthalmia, and urogenital tract inflammation, etc. in colonies and water, soil and grain, which can cause pneumonia, enteritis, meningitis, peritonitis, and urogenital tract inflammation. The increasing number of resistant strains of Klebsiella pneumoniae has brought a severe challenge to the clinical anti infection treatment. Therefore, the study of Klebsiella pneumoniae will lay the foundation for the research and development of new methods and measures for the prevention and treatment of the infection related diseases. The diseased calf viscera of a cattle farm in Yuzhong County, Lanzhou, Gansu Province, was used as the disease material. Through the isolation and identification of the pathogen, it was determined that the epidemic was caused by Klebsiella pneumoniae infection. Based on this, the biological characteristics of the isolated strains were studied. Finally, the preliminary analysis of the bioinformatics was carried out on the genome of the isolated strain. A certain basis was laid on the pathogenesis and immune mechanism of primary bacteria. The research content and results mainly include the separation and identification of the YZ strain of Klebsiella pneumoniae (.1.), based on the clinical comprehensive diagnosis, the isolation of pathogenic bacteria from the sick material, and the pathogenicity test and drug sensitivity test on the basis of purification. Biochemical tests, the amplification and sequence analysis of the 16S rRNA gene, Khe gene and some virulence genes. The results showed that, except for the H2S test, the other biochemical characteristics of the isolated bacteria were all conformed to Klebsiella pneumoniae, and the 16S rRNA gene and the Khe gene sequence were 99% of the homology of all the known sequence of Klebsiella pneumoniae, and three different virulence of the bacteria. The homology of the gene sequence was more than 95% with the other strains of Klebsiella pneumoniae. Based on this, the isolation bacteria was identified as Klebsiella pneumoniae and the biological characteristics of the YZ strain.2. Klebsiella pneumoniae strain YZ strain were identified, and the membrane staining characteristics, growth curve and biofilm form were completed. The results showed that the isolated bacteria could not effectively form a capsule; from the drawn growth curve, we can see that the isolate was in the slow period of 0~4 h, after 4 h and entered the logarithmic period of growth, 10~18 h was a stable period, and after 18 h, it entered the decline period; the qualitative and determination of the biofilm was determined. The results showed that the isolated bacteria could form a certain biofilm, and the median lethal dose of the bacteria was 1.38 * 108 CFU, and the virulence was relatively weak. The vaccine was prepared by different concentrations (1 * 107 cfu/mL~1 x 1010 cfu/mL) after inactivation of the isolated bacteria, and there was no abnormal phenomenon after inoculation of the mice. The toxicity test showed that when the concentration of the vaccine was 1 * 1010 cfu/mL, the protection rate could reach 100%, indicating that the isolated bacteria had a good protective effect. The genomic analysis of the ELISA titer of the antiserum of mice up to more than 1:3 200 of the YZ strain of Klebsiella pneumoniae was to understand the cause and pathogenicity of the capsule defect of the isolated bacteria. The genome length of the genome was completed and the sequencing results were preliminarily analyzed. The results showed that the genome length of the isolated bacteria was 5336251 BP, the content of GC was 57.444%, 120 Scaffold and 166 Contig were contained, and 80 tRNA and 3 rRNA were predicted, and 5205 genes were found in the genome, and found by comparison with the other strains of the genus. The four conserved genes associated with the transfer and surface assembly of capsular polysaccharide are lack of WZB and WZC genes. In addition, only negative regulatory genes, Fur, CRP and SugE, are found in the regulation subsets of different roles in the capsule synthesis, and no rmpA, magA, wabG and other genes are found in the virulence genes, and also in many predictive genes. There are some genes with unknown functions. The above results laid a foundation for further study on the causes of capsule forming defects and elucidating their pathogenic mechanism.

【学位授予单位】：甘肃农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.61

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