【摘要】：沙門菌(Salmonella)可以通過污染食物感染人和動(dòng)物,引起多種人和動(dòng)物的急性或慢性傳染病,是一種在公共衛(wèi)生學(xué)上具有重要意義的人畜共患病原菌。在世界各地的食物中毒病例中,沙門菌引起的中毒病例占首位或第二位,嚴(yán)重威脅著人類的身體健康和畜牧業(yè)的健康發(fā)展。目前許多發(fā)達(dá)國家諸如美國都已建立了相對(duì)完善的沙門菌流行病學(xué)監(jiān)測和耐藥監(jiān)控體系,流行病學(xué)數(shù)據(jù)較為豐富,同時(shí)也開展了較深入的耐藥機(jī)制研究,而我國在這方面的研究還比較滯后。為了了解動(dòng)物沙門菌的流行情況和藥物敏感性,以及對(duì)氟苯尼考的耐藥機(jī)制。本研究對(duì)臨床上疑似沙門菌病的病料進(jìn)行病原分離和細(xì)菌的多重PCR鑒定,選取臨床分離株S101022、0605E2-H,并針對(duì)floR、 AcrB兩個(gè)耐藥基因,采用λ-Red同源重組法構(gòu)建相應(yīng)的基因缺失株,初步探討了這兩個(gè)耐藥基因與氟苯尼考耐藥的關(guān)系,為進(jìn)一步深入研究沙門菌氟苯尼考耐藥的分子機(jī)制奠定了基礎(chǔ)。1.沙門菌的分離鑒定及耐藥性分析對(duì)臨床上疑似沙門菌病的病料進(jìn)行病原分離和細(xì)菌的多重PCR鑒定,共分離到沙門菌61株,其中腸炎沙門菌10株,雞白痢沙門菌12株,鼠傷寒沙門菌39株,月份分布顯示,7-8月分離率最高。K-B法測定分離株對(duì)23種抗菌藥物的敏感性以及部分藥物的MIC結(jié)果顯示,動(dòng)物源沙門菌耐藥性總體呈現(xiàn)上升趨勢,所有菌株對(duì)青霉素、紅霉素、萬古霉素耐藥,90.16%對(duì)6種及其以上抗菌藥耐藥。選擇氟苯尼考耐藥菌株擴(kuò)增floR、fexA、fexB等基因,結(jié)果顯示,8株攜帶floR,其他基因未檢測到,對(duì)克隆的floR基因的核苷酸序列進(jìn)行分析,與Genbank中已報(bào)道動(dòng)物源性沙門菌floR基因核苷酸序列比較,發(fā)現(xiàn)同源性在99.3%-100%,氨基酸序列的同源性在99.2%-100%,分別在147、160、228、293位發(fā)生了氨基酸的替代。2.氟苯尼考相關(guān)耐藥基因缺失株的構(gòu)建及其耐藥表達(dá)研究選取都柏林沙門菌S101022、鼠傷寒沙門菌0605E2-H,針對(duì)不同耐藥基因floR、 AcrB,使用λ-Red同源重組法,構(gòu)建6個(gè)耐藥基因缺失株,PCR擴(kuò)增以及突變株序列測序結(jié)果顯示6個(gè)基因突變株構(gòu)建成功。采用第一章中的方法測定相應(yīng)缺失株的藥物敏感性,其結(jié)果顯示缺失株對(duì)氟苯尼考的耐藥性成功丟失。細(xì)菌的生長曲線測定結(jié)果顯示,雙基因缺失株的生長速度略慢于野生株,其余菌株的生長速度并無明顯差異。采用Q-PCR方法對(duì)floR.AcrB耐藥基因的表達(dá)進(jìn)行了測量,結(jié)果顯示,floR缺失株無論是否氟苯尼考的選擇壓力下,AcrB的表達(dá)量會(huì)呈現(xiàn)不同程度的增加,添加CCCP后,AcrB的表達(dá)量并沒有顯著差異；而AcrB缺失株在CCCP的選擇性壓力下,floR的表達(dá)量卻有了一定程度的升高,證明AcrB的表達(dá)對(duì)外界環(huán)境改變(尤其是抗生素)有著相關(guān)程度的依賴性,而正常環(huán)境下floR的表達(dá)并沒有顯著的差異；AcrB和floR在氟苯尼考耐藥過程中的表達(dá)差異并不明顯。3.HPLC法測定氟苯尼考在耐藥和基因缺失株內(nèi)聚集的研究采用HPLC法測定野生株S101022、0605E2-H和基因缺失株S101022△floR. S101022△AcrB.0605E2-H△f1oR、0605E2-H△AcrB攝取氟苯尼考的差異,結(jié)果顯示,與floR和AcrB基因缺失株相比,耐藥的野生株S101022和0605E2-H對(duì)氟苯尼考的攝取量明顯較少,而在加入氟苯尼考的10 min,野生株的氟苯尼考的積聚量約是雙基因缺失株的1/2。而在耐藥過程中加入CCCP和PAβN(一種外排泵抑制劑),發(fā)現(xiàn)CCCP對(duì)△AcrB氟苯尼考的積聚量影響較大,對(duì)△floR影響并不顯著,而PAβN對(duì)△AcrB氟苯尼考的積聚量影響較小,而對(duì)△floR影響及其顯著,可見CCCP可能主要針對(duì)的是floR基因介導(dǎo)的泵出系統(tǒng),而PAβN主要針對(duì)的是AcrB介導(dǎo)的泵出系統(tǒng),兩者對(duì)氟苯尼考的能量抑制存在特異性,且在氟苯尼考的耐藥過程中均發(fā)揮了一定的作用。
[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.
【學(xué)位授予單位】：揚(yáng)州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S852.61

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