本文選題：金黃色葡萄球菌 + 共抗性��； 參考：《蘭州大學(xué)》2016年博士論文

【摘要】：抗生素作為人類20世紀(jì)最偉大的發(fā)明之一,在醫(yī)療發(fā)展史上具有里程碑式的意義。它的出現(xiàn)挽救了無數(shù)人的生命,讓許多患者免除病痛的折磨。截至目前使用在醫(yī)療衛(wèi)生活動(dòng)中的抗生素已有千余種,其中的許多種還被大量用于農(nóng)業(yè)、畜牧和水產(chǎn)養(yǎng)殖過程中以提高產(chǎn)量。在上世紀(jì)60年代以MRSA(Methicillin-resistant Staphylococcus aureus)菌株的出現(xiàn)為標(biāo)志,一系列廣譜耐藥的超級(jí)細(xì)菌引起了人們的恐慌,如NDM-1,VRE(Vancomycin-resistant Enterococci)和CA-MASA等。濫用抗生素曾經(jīng)被認(rèn)為是造成這些超級(jí)耐藥細(xì)菌的罪魁禍?zhǔn)?但隨后限制抗生素的使用以后并沒有減少或延緩廣譜抗藥病菌的出現(xiàn)速度。同時(shí)研究發(fā)現(xiàn)抗生素抗性與重金屬或者其它抗菌劑抗性之間存在關(guān)聯(lián)。環(huán)境中的重金屬可以迫使微生物保持其金屬抗性,而抗生素抗性也會(huì)同時(shí)保存下來。由于在自然環(huán)境中金屬的存在時(shí)間遠(yuǎn)遠(yuǎn)長于抗生素,由金屬誘導(dǎo)和保持的抗生素抗性在環(huán)境中會(huì)逐漸富集,而如果這些抗性菌引起疾病就會(huì)面臨無藥可用的局面,并演變成為一場災(zāi)難。因此,研究微生物尤其是病原微生物對(duì)于重金屬和抗生素的共抗性機(jī)制是十分重要的,有助于尋找減緩抗生素抗性擴(kuò)散的途徑。在本實(shí)驗(yàn)中從黃河受工業(yè)廢水污染的淤泥中分離出了一株具有較高的重金屬抗性的菌株,該菌株對(duì)金屬鉻(Cr)、鉛(Pb)和錳(Mn)的MIC分別達(dá)到了6 m M,1 m M和6 m M。該菌株同時(shí)還有較高的抗生素抗性,對(duì)氨芐青霉素(Amp)和氯霉素(Chlo)的MIC分別達(dá)到了0.51 mg/m L和0.25 mg/m L。同時(shí)還發(fā)現(xiàn)該菌株的重金屬抗性和抗生素抗性之間可以相互誘導(dǎo),當(dāng)使用0.50 m M的鉻(Cr)或者錳(Mn)誘導(dǎo)之后,菌株對(duì)氨芐的抗性水平分別達(dá)到了2.5 mg/m L和1.6 mg/m L。當(dāng)時(shí)用0.15 mg/m L的Amp或者0.05 mg/m L Chlo誘導(dǎo)之后,該菌株對(duì)鉻(Cr)的抗性水平分別達(dá)到了10 m M和8 m M。另外,當(dāng)重金屬和抗生素同時(shí)出現(xiàn)時(shí),菌株對(duì)重金屬和抗生素的抗性都會(huì)提高。在培養(yǎng)基中鉻(Cr)的濃度達(dá)到6 m M時(shí),菌株同時(shí)對(duì)氨芐青霉素(Amp)的抗性可以達(dá)到0.75 mg/m L。通過對(duì)該菌株核糖體16S序列的分析,表明該菌株為金黃色葡萄球菌的一個(gè)亞種,我們將其命名為Staphylococcus aureus LZ-01。為了尋找S aureus LZ-01菌株對(duì)重金屬和抗生素共抗性的機(jī)制,我們首先通過轉(zhuǎn)錄組測序的方法分析了可以響應(yīng)金屬鉻壓力的基因。結(jié)果表明在可預(yù)測和注釋的2370個(gè)基因中有512個(gè)基因的表達(dá)明顯上調(diào)。通過這些上調(diào)基因的功能分析表明S.aureus LZ-01菌株對(duì)鉻的抗性途徑可能有三條,包括胞外還原、胞內(nèi)還原和外排作用,其中還原途徑對(duì)抗性的貢獻(xiàn)較弱,外排作用為主要抗性機(jī)制。然后在這些上調(diào)基因中篩選出了43個(gè)與抗生素抗性相關(guān)的基因,并對(duì)這些基因使用q RT-PCR的方法進(jìn)行驗(yàn)證。在添加氨芐青霉素壓力的情況下,檢測的43個(gè)基因中僅有少數(shù)幾個(gè)上調(diào)表達(dá),結(jié)合生物信息學(xué)方法,最后確定操縱子emr AB為S.aureus LZ-01菌株對(duì)重金屬和抗生素共抗性的關(guān)鍵基因。利用基因敲除和回補(bǔ)的方法,在實(shí)驗(yàn)中驗(yàn)證了emr A/B基因?qū).aureus LZ-01菌株金屬和抗生素共抗性的貢獻(xiàn)�；騟mr A敲除后菌株對(duì)鉻(Cr)和氨芐青霉素(Amp)的抗性平均下降了80%,而emr B基因敲除后菌株對(duì)鉻(Cr)的抗性下降了30%,對(duì)氨芐青霉素(Amp)的抗性下降了60%。emr A/B基因的敲除會(huì)使菌株喪失金屬和抗生素之間的誘導(dǎo)抗性和交叉抗性。實(shí)驗(yàn)結(jié)果表明emr A/B基因?qū)τ赟.aureus LZ-01菌株金屬和抗生素共抗性十分重要,這種共抗性主要基于三種途徑:一是外排泵作用,二是生物膜作用,三是調(diào)控作用。隨后我們對(duì)emr A/B基因的調(diào)控以及Emr A/B蛋白的結(jié)構(gòu)做了初步分析,研究表明存在一個(gè)二元調(diào)控系統(tǒng),可以感受鉻(Cr)和氨芐青霉素(Amp)的壓力,并在emr A/B基因的啟動(dòng)子區(qū)對(duì)基因轉(zhuǎn)錄進(jìn)行調(diào)控。Emr A/B蛋白結(jié)構(gòu)的初步分析表明這兩個(gè)蛋白可以形成復(fù)合體結(jié)構(gòu),構(gòu)成一個(gè)外排泵。本實(shí)驗(yàn)使用具有臨床價(jià)值的金黃色葡萄球菌作為研究對(duì)象,深入研究了該菌株產(chǎn)生金屬和抗生素共抗性的機(jī)制。并且首次發(fā)掘了emr A/B基因在革蘭氏陽性菌中的功能,提出了產(chǎn)生共抗性的具體途徑。本實(shí)驗(yàn)的結(jié)果對(duì)于控制抗生素抗性基因的擴(kuò)散、設(shè)計(jì)新型抗菌藥物都有一定的借鑒意義,同時(shí)為消除環(huán)境污染、評(píng)估污染生態(tài)危害提供科學(xué)支撐和理論依據(jù)。
[Abstract]:As one of the greatest inventions of mankind in the twentieth Century, antibiotics have a milestone in the history of medical development. The emergence of antibiotics has saved the lives of countless people and exempted many patients from the pain. As of now, there are more than a thousand antibiotics used in medical and health activities, many of which are also used in agriculture and animal husbandry. And in the process of aquaculture to increase production. A series of broad-spectrum resistant superbacteria caused panic, such as NDM-1, VRE (Vancomycin-resistant Enterococci) and CA-MASA, as marked by the emergence of MRSA (Methicillin-resistant Staphylococcus aureus) strain in the 60s of last century. This is the culprit of these super resistant bacteria, but subsequent restrictions on the use of antibiotics did not reduce or postpone the emergence of broad-spectrum antifungal pathogens. As the existence of metals in the natural environment is much longer than that of antibiotics, the resistance of metals induced and maintained is gradually enriched in the environment, and if these resistant bacteria cause disease, it will face the situation of non drug availability and become a disaster. Therefore, the study of microorganisms is especially important. It is important that the co resistance mechanism of pathogenic microbes to heavy metals and antibiotics is very important and helps to find a way to slow the diffusion of antibiotic resistance. In this experiment, a strain with high heavy metal resistance was isolated from the sludge contaminated by industrial wastewater in the Yellow River. This strain has a M of metal chromium (Cr), lead (Pb) and manganese (Mn). IC reached 6 m M, 1 m M and 6 m M., which had high antibiotic resistance. The MIC of ampicillin (Amp) and chloramphenicol (Chlo) reached 0.51 mg/m L and 0.25 respectively. After the strain, the resistance level of the strain to ampicin reached 2.5 mg/m L and 1.6 mg/m L., respectively, when the 0.15 mg/m L Amp or 0.05 mg/m L Chlo were induced. The resistance level of the strain to chromium (Cr) reached 10 m and 8 respectively. When the concentration of chromium (Cr) reached 6 m M in the medium, the resistance of the strain to ampicillin (Amp) could reach 0.75 mg/m L. through the analysis of the ribosome 16S sequence of the strain, indicating that the strain was a subspecies of Staphylococcus aureus, and we named it Staphylococcus aureus LZ-01. in order to find S aureus. The mechanism of strain resistance to heavy metals and antibiotics, we first analyzed the genes that could respond to metal chromium pressure by a transcriptional sequencing method. The results showed that 512 of the 2370 genes in the predicable and annotated genes were obviously up-regulated. Through the power analysis of these up - regulated genes, the S.aureus LZ-01 strain was shown to chromium. There are three pathways in the resistance pathway, including extracellular reduction, intracellular reduction and exocytosis, in which the contribution of the reductive pathway is weak and the efflux is the main resistance mechanism. Then, 43 genes related to antibiotic resistance are screened out in these up-regulated genes, and the Q RT-PCR method is used to verify these genes. In the case of benzylpenicillin pressure, only a few of the 43 genes were up-regulated, combined with bioinformatics methods, the operon EMR AB was finally identified as the key gene for the co resistance of S.aureus LZ-01 to heavy metals and antibiotics. The EMR A/B gene was verified by the method of gene knockout and supplementation. The EMR A/B gene was verified to be S.aureus LZ-0 in the experiment. The contribution of 1 strain metal and antibiotic co resistance. After gene EMR A knockout, the resistance to chromium (Cr) and ampicillin (Amp) decreased by an average of 80%, while the resistance to chromium (Cr) decreased by 30% after the EMR B knockout, and the decrease of the resistance to ampicillin (Amp) by 60%.emr A/B gene knockout would cause the strain to lose metal and antibiotics The experimental results show that the EMR A/B gene is very important for the resistance of the S.aureus LZ-01 strain metal and antibiotic resistance. This co resistance is mainly based on three ways: one is the efflux pump, the two is the biofilm action, and the three is the regulatory effect. Then, we regulate the EMR A/B gene and the structure of Emr A/B protein. A preliminary analysis shows that there is a two element control system that can feel the pressure of chromium (Cr) and ampicillin (Amp), and a preliminary analysis of the structure of.Emr A/B protein in the promoter region of the EMR A/B gene indicates that the two proteins can form a complex structure and form an outer row pump. The clinical value of Staphylococcus aureus was studied, and the mechanism of producing resistance to antibiotics was studied. The function of EMR A/B gene in Gram-positive bacteria was discovered for the first time, and the specific way to produce CO resistance was proposed. The results of this experiment were set up to control the spread of antibiotic resistance genes. It is of certain reference significance for new antibacterial agents to be calculated, and it also provides scientific support and theoretical basis for eliminating environmental pollution and assessing the ecological hazards of pollution.

【學(xué)位授予單位】：蘭州大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：X703;X172
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本文編號(hào)：1824669