【摘要】： 可自主轉(zhuǎn)移質(zhì)粒在環(huán)境中的接合轉(zhuǎn)移對細菌耐藥性的傳播起著重要作用。耐藥細菌可通過食物鏈進入人體,從而加劇細菌耐藥性問題。納米材料和良好的應(yīng)用前景,其廣泛使用必然會導(dǎo)致水環(huán)境中的納米材料增加。因其獨特的物理化學(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,可能會影響質(zhì)粒的接合轉(zhuǎn)移,目前還沒有相關(guān)報道。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RP4接合轉(zhuǎn)移的影響機制。 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小時后,0.005~5mmol/L納米氧化鋁對轉(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,5mmol/L和50mmol/L納米Al2O3組接合率分別為空白對照組的150倍和40倍,與空白對照組相比具有非常顯著的差異。TEM的觀察結(jié)果顯示接合細菌的細胞外膜互相融合形成致密電子帶�？瞻讓φ战M雖然能觀察到許多細菌緊密接觸,但只有單個供、受體菌形成接合連接,5mmol/L和50mmol/L納米Al2O3組能觀察到多個細菌同時發(fā)生接合。 第二部份探討了納米氧化鋁對接合轉(zhuǎn)移的影響與接合時間、接合細菌的初始濃度、溫度和pH值的關(guān)系。接合菌濃度105cfu/ml時,無論是否有納米氧化鋁干預(yù),轉(zhuǎn)接合子數(shù)始終隨時間的延長而增加,0.5mmol/L Al2O3能縮短轉(zhuǎn)接合子出現(xiàn)的時間,并且在接合時間內(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,納米材料對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,50mmol/L Al2O3能非常顯著地提高轉(zhuǎn)接合子數(shù)(P0.001),其它菌液濃度時對接合無影響;5mmol/LAl2O3始終非常顯著地促進RP4接合轉(zhuǎn)移,轉(zhuǎn)接合子數(shù)分別為相應(yīng)空白對照組的100~400倍(P0.001),在所有的組中促進作用最為顯著;0.5mmol/L Al2O3能顯著促進接合液濃度為106cfu/mL和107cfu/ml時的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)對接合轉(zhuǎn)移率無顯著影響。 為研究納米氧化鋁對RP4接合的促進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菌中的分布情況及對細菌超微結(jié)構(gòu)的影響,并檢測細菌抗氧化系統(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菌胞內(nèi)沉積,5mmol/L納米Al2O3組細菌胞質(zhì)凝聚,而50mmol/L Al2O3組部份細菌觀察不到完整的細胞壁和細胞膜結(jié)構(gòu)。檢測0.005~5mmol/L納米氧化鋁干預(yù)對細菌抗氧化酶系統(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照組,其它組則無顯著差異。 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。納米氧化鋁可能會影響接合基因的表達。采用啟動子融合技術(shù),構(gòu)建trbBp-lacZ和trfAp-lacZ轉(zhuǎn)錄融合子,觀察納米Al2O3干預(yù)對接合配接對形成系統(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照組,5mmol/L和50mmol/L納米Al2O3組之間β-半乳糖苷酶活力無顯著差異。 接合基因的表達受整體調(diào)節(jié)因子調(diào)控,應(yīng)用實時熒光定量PCR的方法檢測整體調(diào)控因子korA、korB和trbA mRNA的表達水平,觀察0.05~5mmol/L納米Al2O3干預(yù)對整體調(diào)控因子korA、korB和trbA mRNA表達的影響,結(jié)果顯示與空白對照組相比,5mmol/L和50mmol/L納米氧化鋁作用后korB和trbA的表達量均顯著上升,korA mRNA的表達水平與空白對照組具有上升趨勢,但是沒有顯著差異。5mmol/L和50mmol/L納米氧化鋁間各基因的mRNA表達量也沒有顯著差異。 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)對蛋白質(zhì)譜表達的影響,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個在5mmol/L納米氧化鋁組表達上調(diào)的蛋白進行質(zhì)譜分析,成功地鑒定了3個蛋白。其中2個蛋白為供體菌E.coli HB101表達,分別為蘋果酸合酶G和丙酮酸激酶,另一個蛋白為受體菌MS1表達,是一種可能的過氧化物酶。 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)境中細菌的耐藥水平。納米Al2O3對轉(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),損傷細菌的細胞膜和細胞壁結(jié)構(gòu)。氧化鋁對細菌細胞膜結(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)細胞膜嚴重損傷時則不利于接合進行。 3.納米氧化鋁能促進整體調(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,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,該過程需啟動許多基因表達,消耗大量能量,因此促進供體菌中能量代謝相關(guān)的酶表達升高。受體菌中產(chǎn)生一種未知的過氧化物酶,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理,提出了納米材料可能會促進細菌耐藥性在水環(huán)境中的傳播,危害水環(huán)境安全,并有可能促進社區(qū)獲得性病原菌的耐藥性。該研究豐富了納米材料對健康風(fēng)險理論,為納米材料的污染防護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：2286919