沙门菌的分离鉴定及其氟苯尼考耐药机制的研究

发布时间：2018-08-13 16:07

【摘要】：沙门菌(Salmonella)可以通过污染食物感染人和动物,引起多种人和动物的急性或慢性传染病,是一种在公共卫生学上具有重要意义的人畜共患病原菌。在世界各地的食物中毒病例中,沙门菌引起的中毒病例占首位或第二位,严重威胁着人类的身体健康和畜牧业的健康发展。目前许多发达国家诸如美国都已建立了相对完善的沙门菌流行病学监测和耐药监控体系,流行病学数据较为丰富,同时也开展了较深入的耐药机制研究,而我国在这方面的研究还比较滞后。为了了解动物沙门菌的流行情况和药物敏感性,以及对氟苯尼考的耐药机制。本研究对临床上疑似沙门菌病的病料进行病原分离和细菌的多重PCR鉴定,选取临床分离株S101022、0605E2-H,并针对floR、 AcrB两个耐药基因,采用λ-Red同源重组法构建相应的基因缺失株,初步探讨了这两个耐药基因与氟苯尼考耐药的关系,为进一步深入研究沙门菌氟苯尼考耐药的分子机制奠定了基础。1.沙门菌的分离鉴定及耐药性分析对临床上疑似沙门菌病的病料进行病原分离和细菌的多重PCR鉴定,共分离到沙门菌61株,其中肠炎沙门菌10株,鸡白痢沙门菌12株,鼠伤寒沙门菌39株,月份分布显示,7-8月分离率最高。K-B法测定分离株对23种抗菌药物的敏感性以及部分药物的MIC结果显示,动物源沙门菌耐药性总体呈现上升趋势,所有菌株对青霉素、红霉素、万古霉素耐药,90.16%对6种及其以上抗菌药耐药。选择氟苯尼考耐药菌株扩增floR、fexA、fexB等基因,结果显示,8株携带floR,其他基因未检测到,对克隆的floR基因的核苷酸序列进行分析,与Genbank中已报道动物源性沙门菌floR基因核苷酸序列比较,发现同源性在99.3%-100%,氨基酸序列的同源性在99.2%-100%,分别在147、160、228、293位发生了氨基酸的替代。2.氟苯尼考相关耐药基因缺失株的构建及其耐药表达研究选取都柏林沙门菌S101022、鼠伤寒沙门菌0605E2-H,针对不同耐药基因floR、 AcrB,使用λ-Red同源重组法,构建6个耐药基因缺失株,PCR扩增以及突变株序列测序结果显示6个基因突变株构建成功。采用第一章中的方法测定相应缺失株的药物敏感性,其结果显示缺失株对氟苯尼考的耐药性成功丢失。细菌的生长曲线测定结果显示,双基因缺失株的生长速度略慢于野生株,其余菌株的生长速度并无明显差异。采用Q-PCR方法对floR.AcrB耐药基因的表达进行了测量,结果显示,floR缺失株无论是否氟苯尼考的选择压力下,AcrB的表达量会呈现不同程度的增加,添加CCCP后,AcrB的表达量并没有显著差异；而AcrB缺失株在CCCP的选择性压力下,floR的表达量却有了一定程度的升高,证明AcrB的表达对外界环境改变(尤其是抗生素)有着相关程度的依赖性,而正常环境下floR的表达并没有显著的差异；AcrB和floR在氟苯尼考耐药过程中的表达差异并不明显。3.HPLC法测定氟苯尼考在耐药和基因缺失株内聚集的研究采用HPLC法测定野生株S101022、0605E2-H和基因缺失株S101022△floR. S101022△AcrB.0605E2-H△f1oR、0605E2-H△AcrB摄取氟苯尼考的差异,结果显示,与floR和AcrB基因缺失株相比,耐药的野生株S101022和0605E2-H对氟苯尼考的摄取量明显较少,而在加入氟苯尼考的10 min,野生株的氟苯尼考的积聚量约是双基因缺失株的1/2。而在耐药过程中加入CCCP和PAβN(一种外排泵抑制剂),发现CCCP对△AcrB氟苯尼考的积聚量影响较大,对△floR影响并不显著,而PAβN对△AcrB氟苯尼考的积聚量影响较小,而对△floR影响及其显著,可见CCCP可能主要针对的是floR基因介导的泵出系统,而PAβN主要针对的是AcrB介导的泵出系统,两者对氟苯尼考的能量抑制存在特异性,且在氟苯尼考的耐药过程中均发挥了一定的作用。
[Abstract]:Salmonella can infect humans and animals by contaminating food, causing acute or chronic infectious diseases in many humans and animals. Salmonella is a zoonotic pathogen of great importance in public health. At present, many developed countries, such as the United States, have established relatively perfect epidemiological surveillance and drug resistance surveillance systems for Salmonella. The epidemiological data are relatively rich, and at the same time, a deeper study on drug resistance mechanism has been carried out. Epidemiology and drug susceptibility of Salmonella in animals, and the mechanism of resistance to florfenicol. In this study, pathogen isolation and multiplex PCR identification of clinical suspected Salmonella were carried out. Clinical isolates S101022,0605E2-H were selected, and two resistant genes, floR and AcrB, were constructed by lambda-Red homologous recombination method. The relationship between the two drug-resistant genes and the resistance of flubenicol was preliminarily discussed, which laid a foundation for further study on the molecular mechanism of flubenicol resistance of Salmonella. 1. Isolation and identification of Salmonella and analysis of drug resistance were carried out to isolate pathogens from clinical suspected Salmonella diseases and identify bacteria by multiplex PCR. Sixty-one strains of Salmonella were isolated, including 10 strains of Salmonella enteritidis, 12 strains of Salmonella pullorum and 39 strains of Salmonella typhimurium. The monthly distribution showed that the isolation rate was the highest from July to August. Sensitivity of the isolates to 23 antimicrobial agents and MIC of some antimicrobial agents were determined by K-B method. Resistance to penicillin, erythromycin and vancomycin was 90.16%. The floR, fexA and fexB genes were amplified from flufenicol-resistant strains. The results showed that 8 strains carried floR and other genes were not detected. The nucleotide sequence of the cloned floR gene was analyzed and compared with the floR gene of Salmonella from animal origin reported in Genbank. Nucleotide sequence comparison showed that the homology was 99.3%-100%, amino acid sequence homology was 99.2%-100%, and amino acid substitution occurred at 147,160,228,293 sites respectively. Six resistant gene deletion strains were constructed by lambda-Red homologous recombination. PCR amplification and sequencing of the mutants showed that the mutants were successfully constructed. Drug susceptibility of the corresponding deleted strains was determined by the method in Chapter 1. The results showed that the resistance of the deleted strains to florfenicol was successfully lost. The results of growth curve showed that the growth rate of the two-gene deleted strains was slightly slower than that of the wild strains, and there was no significant difference in the growth rate of the other strains. The expression of AcrB did not differ significantly after CCCP addition, but the expression of floR in AcrB deletion strains increased to a certain extent under CCCP selective pressure, which proved that the expression of AcrB was dependent on environmental changes (especially antibiotics), while the expression of floR in normal environment was not significant. The difference of expression of AcrB and floR in the process of resistance to florfenicol was not significant. 3. Determination of florfenicol concentration in drug-resistant and gene-deleted strains by HPLC. Determination of the difference of uptake of florfenicol by wild strain S101022,0605E2-H and gene-deleted strain S101022 Delta floR.S101022 Delta AcrB.0605E2-H Delta AcrB by HPLC The results showed that compared with floR and ACrB gene deletion strains, the uptake of florfenicol in resistant wild strains S10102 2 and 0605E2-H was significantly lower, while the accumulation of florfenicol in wild strains was about 1/2 of that in double gene deletion strains at 10 min after adding florfenicol. At present, CCCP has a great influence on the accumulation of AcrB flubenicol, but not on floR. PA beta N has a small effect on the accumulation of AcrB flubenicol, but has a significant effect on floR. It is clear that CCCP may mainly aim at the floR gene-mediated pumping system, while PA beta N mainly aims at the AcrB-mediated pumping system. The energy inhibition of florfenicol is specific and plays a certain role in the process of resistance to florfenicol.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.61

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