發(fā)布時(shí)間：2018-10-22 10:36

【摘要】： 可自主轉(zhuǎn)移質(zhì)粒在環(huán)境中的接合轉(zhuǎn)移對(duì)細(xì)菌耐藥性的傳播起著重要作用。耐藥細(xì)菌可通過(guò)食物鏈進(jìn)入人體,從而加劇細(xì)菌耐藥性問(wèn)題。納米材料和良好的應(yīng)用前景,其廣泛使用必然會(huì)導(dǎo)致水環(huán)境中的納米材料增加。因其獨(dú)特的物理化學(xué)特性,如小尺寸和大比表面積等,納米材料具有很高的反應(yīng)活性,可能會(huì)影響質(zhì)粒的接合轉(zhuǎn)移,目前還沒(méi)有相關(guān)報(bào)道。本工作以攜帶多重耐藥接合型質(zhì)粒RP4的E.coli HB101為供體菌,Salmonella aberdeen Kauffmann 50312為受體菌,研究了納米氧化鋁對(duì)液相接合條件下RP4接合轉(zhuǎn)移的影響及影響規(guī)律,采用形態(tài)學(xué)、生物化學(xué)、分子生物學(xué)以及蛋白質(zhì)組學(xué)的方法和手段,探討納米氧化鋁對(duì)RP4接合轉(zhuǎn)移的影響機(jī)制。 本工作首先探討了供、受體菌菌液濃度約為109cfu/mL(濃度比為1:3),接合8小時(shí)后,0.005~5mmol/L納米氧化鋁對(duì)轉(zhuǎn)接合子數(shù)的影響。結(jié)果表明RP4的接合轉(zhuǎn)移隨著納米Al2O3濃度的升高具有上升趨勢(shì),5mmol/L和50mmol/L納米Al2O3組接合率分別為空白對(duì)照組的150倍和40倍,與空白對(duì)照組相比具有非常顯著的差異。TEM的觀察結(jié)果顯示接合細(xì)菌的細(xì)胞外膜互相融合形成致密電子帶�？瞻讓�(duì)照組雖然能觀察到許多細(xì)菌緊密接觸,但只有單個(gè)供、受體菌形成接合連接,5mmol/L和50mmol/L納米Al2O3組能觀察到多個(gè)細(xì)菌同時(shí)發(fā)生接合。 第二部份探討了納米氧化鋁對(duì)接合轉(zhuǎn)移的影響與接合時(shí)間、接合細(xì)菌的初始濃度、溫度和pH值的關(guān)系。接合菌濃度105cfu/ml時(shí),無(wú)論是否有納米氧化鋁干預(yù),轉(zhuǎn)接合子數(shù)始終隨時(shí)間的延長(zhǎng)而增加,0.5mmol/L Al2O3能縮短轉(zhuǎn)接合子出現(xiàn)的時(shí)間,并且在接合時(shí)間內(nèi)(90h)轉(zhuǎn)接合子數(shù)增加的頻率始終非常顯著高于空白對(duì)照組,而5mmol/L和50mmol/L納米氧化鋁始終抑制接合。接合菌液濃度升高至106~108cfu/ml,接合8h時(shí),納米材料對(duì)接合的影響與初始接合菌液和納米材料的濃度均相關(guān)。當(dāng)接合菌濃度為108cfu/ml時(shí),50mmol/L Al2O3能非常顯著地提高轉(zhuǎn)接合子數(shù)(P0.001),其它菌液濃度時(shí)對(duì)接合無(wú)影響;5mmol/LAl2O3始終非常顯著地促進(jìn)RP4接合轉(zhuǎn)移,轉(zhuǎn)接合子數(shù)分別為相應(yīng)空白對(duì)照組的100~400倍(P0.001),在所有的組中促進(jìn)作用最為顯著;0.5mmol/L Al2O3能顯著促進(jìn)接合液濃度為106cfu/mL和107cfu/ml時(shí)的RP4的接合轉(zhuǎn)移(P0.05)。溫度(15℃、20℃、25℃、30℃、35℃)和pH值(6.0, 6.5, 7.0, 7.5, 8.0)對(duì)接合轉(zhuǎn)移率無(wú)顯著影響。 為研究納米氧化鋁對(duì)RP4接合的促進(jìn)作用與其濃度之間的關(guān)系,采用激光掃描共聚焦顯微鏡(lazer scanning confocal microscope,LSCM)和TEM觀察納米氧化鋁在細(xì)菌中的分布情況及對(duì)細(xì)菌超微結(jié)構(gòu)的影響,并檢測(cè)細(xì)菌抗氧化系統(tǒng)的變化情況。LSCM觀察結(jié)果表明50mmol/L納米Al2O3組熒光強(qiáng)度高于5mmol/L納米Al2O3組。TEM觀察結(jié)果顯示納米氧化鋁能在細(xì)菌胞內(nèi)沉積,5mmol/L納米Al2O3組細(xì)菌胞質(zhì)凝聚,而50mmol/L Al2O3組部份細(xì)菌觀察不到完整的細(xì)胞壁和細(xì)胞膜結(jié)構(gòu)。檢測(cè)0.005~5mmol/L納米氧化鋁干預(yù)對(duì)細(xì)菌抗氧化酶系統(tǒng)的影響,結(jié)果表明5mmol/L和50mmol/L Al2O3干預(yù)后,細(xì)菌的總抗氧化能力(T-AOC)升高,相應(yīng)的抗氧化酶如超氧化物歧化酶(SOD)、過(guò)氧化氫酶(CAT)以及谷胱苷肽還原酶(GR)活力均顯著高于空白對(duì)照組,其它組則無(wú)顯著差異。 IncP質(zhì)粒接合轉(zhuǎn)移需要接合基因的參與,但是接合基因并非組成型表達(dá)。納米氧化鋁可能會(huì)影響接合基因的表達(dá)。采用啟動(dòng)子融合技術(shù),構(gòu)建trbBp-lacZ和trfAp-lacZ轉(zhuǎn)錄融合子,觀察納米Al2O3干預(yù)對(duì)接合配接對(duì)形成系統(tǒng)(mating pair formation,Mpf)和DNA轉(zhuǎn)移及復(fù)制系統(tǒng)(DNA transfer and replication,Dtr)轉(zhuǎn)錄的影響。結(jié)果表明5mmol/L和50mmol/L納米Al2O3組trbBp和trfAp的β-半乳糖苷酶活力顯著高于空白對(duì)照組,5mmol/L和50mmol/L納米Al2O3組之間β-半乳糖苷酶活力無(wú)顯著差異。 接合基因的表達(dá)受整體調(diào)節(jié)因子調(diào)控,應(yīng)用實(shí)時(shí)熒光定量PCR的方法檢測(cè)整體調(diào)控因子korA、korB和trbA mRNA的表達(dá)水平,觀察0.05~5mmol/L納米Al2O3干預(yù)對(duì)整體調(diào)控因子korA、korB和trbA mRNA表達(dá)的影響,結(jié)果顯示與空白對(duì)照組相比,5mmol/L和50mmol/L納米氧化鋁作用后korB和trbA的表達(dá)量均顯著上升,korA mRNA的表達(dá)水平與空白對(duì)照組具有上升趨勢(shì),但是沒(méi)有顯著差異。5mmol/L和50mmol/L納米氧化鋁間各基因的mRNA表達(dá)量也沒(méi)有顯著差異。 采用二維聚丙烯酰胺凝膠電泳(2-D PAGE)及質(zhì)譜技術(shù),分析5mmol/L納米氧化鋁干預(yù)對(duì)蛋白質(zhì)譜表達(dá)的影響,從蛋白水平探討響應(yīng)納米氧化鋁效應(yīng)的蛋白。根據(jù)2D-PAGE結(jié)構(gòu),選取其中6個(gè)在5mmol/L納米氧化鋁組表達(dá)上調(diào)的蛋白進(jìn)行質(zhì)譜分析,成功地鑒定了3個(gè)蛋白。其中2個(gè)蛋白為供體菌E.coli HB101表達(dá),分別為蘋果酸合酶G和丙酮酸激酶,另一個(gè)蛋白為受體菌MS1表達(dá),是一種可能的過(guò)氧化物酶。 上述結(jié)果表明: 1.納米Al2O3能提高RP4介導(dǎo)的接合轉(zhuǎn)移,可能從而提高水環(huán)境中細(xì)菌的耐藥水平。納米Al2O3對(duì)轉(zhuǎn)接合子數(shù)的影響水平與納米Al2O3及接合菌液的濃度有關(guān)。隨著接合菌液濃度降低,能最大地促進(jìn)RP4接合轉(zhuǎn)移的納米Al2O3的濃度也降低。 2.納米氧化鋁能夠誘導(dǎo)產(chǎn)生活性氧自由基(reactive oxygen species ,ROS),損傷細(xì)菌的細(xì)胞膜和細(xì)胞壁結(jié)構(gòu)。氧化鋁對(duì)細(xì)菌細(xì)胞膜結(jié)構(gòu)的輕微損傷作用可能有利于接合的發(fā)生,但是當(dāng)細(xì)胞膜嚴(yán)重?fù)p傷時(shí)則不利于接合進(jìn)行。 3.納米氧化鋁能促進(jìn)整體調(diào)節(jié)因子korB和trbA的轉(zhuǎn)錄,以調(diào)控供體菌和受體菌中trfAp和trbBp的表達(dá),使接合液中Dtr系統(tǒng)和Mpf系統(tǒng)的基因轉(zhuǎn)錄水平均升高,從而促進(jìn)RP4接合轉(zhuǎn)移。 4.納米氧化鋁干預(yù)后RP4接合轉(zhuǎn)移增加,該過(guò)程需啟動(dòng)許多基因表達(dá),消耗大量能量,因此促進(jìn)供體菌中能量代謝相關(guān)的酶表達(dá)升高。受體菌中產(chǎn)生一種未知的過(guò)氧化物酶,該酶可能參與了響應(yīng)納米氧化鋁的氧化脅迫效應(yīng)。 本工作首次提出納米氧化鋁具有促進(jìn)水環(huán)境中耐藥質(zhì)粒接合轉(zhuǎn)移的現(xiàn)象,并且初步探討了其機(jī)理,提出了納米材料可能會(huì)促進(jìn)細(xì)菌耐藥性在水環(huán)境中的傳播,危害水環(huán)境安全,并有可能促進(jìn)社區(qū)獲得性病原菌的耐藥性。該研究豐富了納米材料對(duì)健康風(fēng)險(xiǎn)理論,為納米材料的污染防護(hù)理論提供了理論依據(jù)。
[Abstract]:The transfer of autonomously transferable plasmids in the environment plays an important role in the propagation of bacterial resistance. The drug-resistant bacteria can enter the human body through the food chain, thus increasing the problem of bacterial resistance. Nanomaterials and good application prospects, their widespread use necessarily lead to an increase in nanomaterials in water environments. Due to its unique physical and chemical properties, such as small size and large specific surface area, nano-materials have very high reaction activity, which may affect the conjugation and transfer of plasmids, and there are no relevant reports at present. coli HB101 containing multiple drug-resistant binding plasmid RP4 was donor strain, Salmonella aberdeen Kaufmann 50312 was the acceptor strain, and the effect of nano-alumina on the transfer of RP4 was studied. Molecular biology and proteomics methods and methods were used to investigate the effect mechanism of nano-alumina on the transfer of RP4. At the beginning of this work, the concentration of bacteria liquid for donor and recipient is about 109cfu/ mL (concentration ratio is 1: 3), after 8 hours of conjugation, 0. 005-5mmol/ L nano-alumina is used to transfer the zygotes. The results showed that the bonding transfer of RP4 increased with the increase of the concentration of nano-Al2O3, and the bonding rate of 5mmol/ L and 50mmol/ L nano-Al2O3 groups was 150 times and 40 times of the blank control group, and it was very significant compared with the blank control group. The results of TEM showed that the outer membrane of the jointed bacteria fused with each other to form a dense layer. Although many bacteria were found to be in close contact with the blank control group, only a single donor was used to form a joint connection, and the 5mmol/ L and 50mmol/ L nano-Al2O3 groups were able to observe multiple bacteria at the same time The second part discusses the effect of nano-alumina on bond transfer and bonding time, the initial concentration and temperature of bonding bacteria. When the concentration of the bacteria was 105cfu/ ml, the number of zygotes increased with the prolongation of time, and 0.5mmol/ L Al2O3 could shorten the time when the zygotes appeared, and the frequency of the increase of the number of zygotes in the time of conjugation (90h) was always very significant. Above blank control group, 5mmol/ L and 50mmol/ L nano-oxidation Aluminum always inhibits bonding. When the concentration of jointed bacteria is raised to 106-108cfu/ ml, the effect of nano-materials on bonding is related to the initial bonding bacteria liquid and nano material at 8h. When the concentration of jointed bacteria was 108cfu/ ml, 50mmol/ L Al2O3 could significantly improve the number of zygotes (P0. 001) and the concentration of other bacteria liquid had no effect on the conjugation; 5mmol/ L _ 2O _ 3 always promoted the transfer of RP4, and the number of zygotes was 100 ~ 400 times higher than that of the corresponding blank control group (P <0.05). P0. 001), most significant in all groups; 0. 5mmol/ L Al2O3 significantly contributed to the junction transfer of RP4 when the concentration of the conjugate was 106cfu/ mL and 107cfu/ ml (P0.05). The temperature (15 鈩，

本文編號(hào)：2286919