【摘要】：目的:滯留菌是臨床上慢性感染反復(fù)發(fā)作的重要原因之一,目前發(fā)現(xiàn)多種人類致病菌可形成滯留菌,滯留菌形成機(jī)制十分復(fù)雜,涉及細(xì)菌的毒力、能量代謝等環(huán)節(jié)。本研究將探討傷寒沙門菌(Salmonella enterica serovar Typhi,S.Typhi)是否可形成滯留菌,環(huán)境因素對滯留菌形成的影響,傷寒沙門菌全局調(diào)控因子Fis在滯留菌形成中所發(fā)揮的作用及其調(diào)節(jié)機(jī)制。方法:1.藥敏試驗(yàn)(紙片法):將麥?zhǔn)蠞舛葹?.5的菌液均勻涂布于M-H瓊脂平板,氨芐西林(AMP)、環(huán)丙沙星(CIP)、卡那霉素(Kana)藥敏紙片貼于平板表面,量取抑菌圈直徑。2.最小抑菌濃度測定:于96孔培養(yǎng)板中將氨芐西林和環(huán)丙沙星倍比稀釋至不同濃度,每孔中加入麥?zhǔn)蠞舛葹?.5的菌液100μL,24 h后能抑制細(xì)菌生長的最低藥物濃度即為該菌株的最小抑菌濃度(MIC)。3.細(xì)菌藥物存活曲線:傷寒沙門菌野生株培養(yǎng)至對數(shù)早期(OD600≈0.4),加入氨芐西林或環(huán)丙沙星作用,每隔1 h取100μL菌液稀釋涂板計(jì)數(shù)。以藥物作用時(shí)間為橫軸,每個(gè)時(shí)間點(diǎn)菌落數(shù)為縱軸,繪制細(xì)菌在不同藥物中的存活曲線。4.細(xì)菌滯留能力檢測:將菌株培養(yǎng)至不同OD600值,加入氨芐西林或者環(huán)丙沙星作用5 h,取100μL菌液稀釋后涂板計(jì)數(shù)。滯留率為5 h菌落數(shù)與未加入藥物之前菌落數(shù)之比。5.q RT-PCR檢測基因表達(dá):傷寒沙門菌野生株、Δfis培養(yǎng)至一定的OD600值,TRIzol法提取細(xì)菌總RNA,逆轉(zhuǎn)錄后q RT-PCR檢測,分析野生株中fis的表達(dá)情況,野生株和Δfis中谷氨酸運(yùn)體相關(guān)基因,rel A和spo T的表達(dá)。6.RT-PCR驗(yàn)證glt操縱子結(jié)構(gòu):分別以glt I、glt L為上下游,以glt J、glt L為上下游各設(shè)計(jì)兩對引物。傷寒沙門菌野生株培養(yǎng)至對數(shù)期(OD600≈0.4),提取細(xì)菌總RNA,用4個(gè)不同位置的下游引物逆轉(zhuǎn)錄。以獲得的c DNA為模板,4對不同位置上下游引物進(jìn)行PCR,瓊脂糖凝膠電泳。7.傷寒沙門菌谷氨酸轉(zhuǎn)運(yùn)體和fis雙缺陷株及回補(bǔ)株的構(gòu)建:同源重組片段glt與自殺質(zhì)粒p GMB151連接,陽性質(zhì)粒電轉(zhuǎn)至Δfis感受態(tài)構(gòu)建缺陷株(記作ΔgltΔfis)。構(gòu)建p BAD-glt重組質(zhì)粒,陽性重組質(zhì)粒和空質(zhì)粒電轉(zhuǎn)至轉(zhuǎn)化ΔgltΔfis感受態(tài),作為glt回補(bǔ)株(ΔgltΔfis+p BAD-glt)和空質(zhì)粒對照株(ΔgltΔfis+p BAD)。結(jié)果:1.藥敏試驗(yàn)結(jié)果表明,傷寒沙門菌野生株、fis基因缺失株對氨芐西林、環(huán)丙沙星、卡那霉素敏感。2.MIC測定結(jié)果說明,傷寒沙門菌野生株、fis基因缺失株對氨芐西林、環(huán)丙沙星敏感且兩者對同一種藥物敏感度相同。3.細(xì)菌藥物存活曲線表明:氨芐西林、環(huán)丙沙星作用于傷寒沙門菌呈二項(xiàng)殺傷曲線變化,存在部分細(xì)菌穩(wěn)定存活。4.傷寒沙門菌可形成滯留菌且滯留菌的產(chǎn)生與細(xì)菌的培養(yǎng)條件、生長時(shí)期相關(guān)。5.通過q RT-PCR分析:fis在對數(shù)早期表達(dá)量最高;相比野生株,在LB培養(yǎng)基中,Δfis的rel A、spo T表達(dá)量下降,而在特殊M9培養(yǎng)基中,相比野生株,Δfis的rel A、spo T表達(dá)量升高;Δfis中谷氨酸轉(zhuǎn)運(yùn)體相關(guān)基因gln H、gln P、glt I、glt K、glt J及glt S表達(dá)量升高相比野生株。6.相比野生株,fis基因缺失株滯留能力下降。7.相比普通LB培養(yǎng)基中的Δfis滯留率與野生株滯留率之間差異,特殊M9培養(yǎng)基中兩者滯留率的差異縮小。8.成功構(gòu)建傷寒沙門菌谷氨酸轉(zhuǎn)運(yùn)體和fis雙缺陷株(ΔgltΔfis)及谷氨酸轉(zhuǎn)運(yùn)體回補(bǔ)株(ΔgltΔfis+p BAD-glt)。9.相比Δfis,ΔgltΔfis滯留能力升高;與空載體對照株相比,glt回補(bǔ)株(ΔgltΔfis+p BAD-glt)滯留能力降低。結(jié)論:傷寒沙門菌可形成滯留菌且受環(huán)境因素的影響,Fis介導(dǎo)傷寒沙門菌滯留菌的形成是通過調(diào)節(jié)谷氨酸轉(zhuǎn)運(yùn)體相關(guān)基因的表達(dá),促進(jìn)谷氨酸的轉(zhuǎn)運(yùn),改變胞內(nèi)的營養(yǎng)狀態(tài),從而影響滯留菌的形成,為進(jìn)一步探索滯留菌形成的具體機(jī)制提供基礎(chǔ)。
[Abstract]:Objective: The retention of bacteria is one of the important causes of the repeated episodes of chronic infection in the clinic. It is found that a variety of human pathogenic bacteria can form the detention bacteria, the mechanism of the formation of the detention bacteria is very complex, and it is related to the virulence and energy metabolism of the bacteria. The effect of Salmonella typhimurium (S. Typhi, S. Typhi) on the formation of retained bacteria, the effect of environmental factors on the formation of detention bacteria, the role of the global regulatory factor Fis in the formation of detention bacteria and its regulation mechanism will be discussed in this study. Method: 1. The drug sensitivity test (the paper-paper method): The bacterial liquid with the concentration of 0.5 was evenly coated on the surface of the plate, and the diameter of the bacteriostatic ring was taken as the diameter of the bacteriostatic ring. and the minimum inhibitory concentration (MIC) of the strain is the minimum inhibitory concentration (MIC) of the strain. The survival curve of the bacterial drug: the wild strain of Salmonella typhi was cultured to the logarithmic early stage (OD600-0.4), with the action of methicillin or ciprofloxacin, and 100. m u.L of bacterial liquid was used to dilute the coated plate at every 1 h. the time of the drug action is the horizontal axis, the number of the colonies per time point is the vertical axis, and the survival curves of the bacteria in different medicines are drawn. Test of the bacteria retention capacity: culture the strain to different OD600 values, add ampicillin or ciprofloxacin for 5 hours, and dilute the 100 & mu; L bacterial solution and then apply the plate to count. 5. q RT-PCR was used to detect the gene expression: the wild strain of Salmonella typhimurium, the yeast fids was cultured to a certain OD600 value, the total RNA of bacteria was extracted by the TRIzol method, and the expression of fids in the wild strain was analyzed by the reverse transcription q RT-PCR. The expression of glutamate transporter-related genes, rel A and spo T in wild plants and plants was verified by RT-PCR: glt I and glt L were the upstream and downstream of glt I and glt L, and two pairs of primers were designed for upstream and downstream of glt J and glt L. The wild strain of Salmonella typhi was cultured to the logarithmic phase (OD600-0.4), and the total RNA of the bacteria was extracted and reverse transcribed with the downstream primers at four different positions. and using the obtained c DNA as a template, 4 pairs of upstream and downstream primers are subjected to PCR and agarose gel electrophoresis. The construction of the glutamate transporter and the fids double-defective strain of the Salmonella typhi and the repair strain: the homologous recombination fragment glt is connected with the suicide plasmid pGMB151, and the positive plasmid is electrically transferred to the disulfide competent state to construct the defective strain (to be recorded as the yeast glt-fids). The pBAD-glt recombinant plasmid was constructed, and the positive recombinant plasmid and the empty plasmid were transferred to the transformed glt-fids competent state as the glt tfids + p BAD-glt and the empty plasmid control strain (Bglt-fids + p BAD). Results: 1. The results of drug sensitivity test showed that the wild strain of Salmonella typhi and the deletion of fids were sensitive to methicillin, ciprofloxacin and kanamycin. Ciprofloxacin is sensitive and both have the same sensitivity to the same drug. The survival curves of the bacterial drug show that the effect of methicillin and ciprofloxacin on Salmonella typhi is the change of two anti-kill curves, and some of the bacteria live stably. Salmonella typhi can form the detention bacteria and the generation of the detention bacteria is related to the culture conditions and the growth period of the bacteria. Through q-RT-PCR analysis, the expression of fids in the logarithmic early stage was the highest; in the LB medium, the expression of rel A and spo T of the fids decreased, while in the medium of the special M9, the expression of rel A and the spo T of the wild strain and FFIs increased. The expression of gln H, gln P, glt I, glt K, glt J and glt S in Glutamic acid transporter was higher than that of wild strain. Compared with the wild strain, the retention capacity of the fids gene is reduced. The difference between the retention rate and the retention rate of the wild strain in the medium of the normal LB medium was compared with that of the wild strain, and the difference of the retention rate of the two in the M9 medium was reduced. Salmonella typhimurium glutamate transporter and fids double-defective strain (tglt-fids + p BAD-glt) were successfully constructed. The retention capacity of glt-fids + p BAD-glt decreased compared with the empty vector control. Conclusion: Salmonella typhimurium can form the detention bacteria and is affected by the environmental factors, and the formation of the Fis-mediated Salmonella typhi is by regulating the expression of the related gene of the glutamate transporter, promoting the transport of the glutamic acid, changing the nutrient state in the cell, and thus affecting the formation of the detention bacteria, and provides the basis for further exploring the specific mechanism for the formation of the detention bacteria.
【學(xué)位授予單位】：江蘇大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R378

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