本文選題：金黃色葡萄球菌 + 松鼠葡萄球菌�。� 參考：《天津醫(yī)科大學》2007年博士論文

【摘要】： 金黃色葡萄球菌是引起醫(yī)院內(nèi)獲得性感染和社區(qū)獲得性感染的重要致病菌之一。β-內(nèi)酰胺類抗生素曾經(jīng)是治療金黃色葡萄球菌感染的有效武器，但隨著耐甲氧西林的金黃色葡萄球菌(Methicillin resistant Staphylococcus aureus，MRSA)分離率的逐漸增加，金黃色葡萄球菌引起的感染成為臨床上非常棘手的問題。耐藥的主要決定基因為mecA。mecA編碼的青霉素結(jié)合蛋白2A(penicillin-binding protein 2A，PBP2A)對β-內(nèi)酰胺類抗生素親和力很低，當固有的PBP被β-內(nèi)酰胺類抗生素飽和時，PBP2A可替代這些PBPs的轉(zhuǎn)肽功能催化細胞壁合成。mecA基因并非金黃色葡萄球菌的固有基因，而是一外源基因，其來源有很多假設，目前認為是從松鼠葡萄球菌中獲得。大多數(shù)松鼠葡萄球菌的mecA并不提供抗藥性，但把活化的松鼠葡萄球菌mecA引入到敏感的金黃色葡萄球菌可使后者的抗藥性大大增加，松鼠葡萄球菌的mecA基因產(chǎn)物能夠利用金黃色葡萄球菌的細胞壁前體合成金黃色葡萄球菌的細胞壁。最近，我們分離到了既含有松鼠葡萄球菌的mecA又含有金黃色葡萄球菌的mecA的松鼠葡萄球菌K3，K3為黃色菌落，在沒有抗生素存在時能自發(fā)分化為K3y(黃色菌落)和K3w(白色菌落)。前者表型和基因型與親代菌完全相同，后者僅含有松鼠葡萄球菌mecA而丟失了金黃色葡萄球菌的mecA。本論文將針對這兩個mecA基因在松鼠葡萄球菌的耐藥性及細胞壁合成中的作用進行研究。 目的：進一步研究松鼠葡萄球菌的生物學特征和基因特征以期更深入的理解金黃色葡萄球菌耐藥性基因mecA來源以及MRSA菌株的耐藥機制；同時，認識臨床上分離率日益增加的松鼠葡萄球菌的耐藥機制。方法：通過抗生素壓力選擇出松鼠葡萄球菌K3的高度均一耐藥株；應用E-test和PAP試驗來檢測松鼠葡萄球菌對苯唑西林的耐藥性；PCR擴增兩個mecA基因并制備DNA探針；多重PCR用于菌株K3和K8的SCCmec分型；應用Northern blot和Western blot分別檢測兩個mecA基因在轉(zhuǎn)錄水平和蛋白質(zhì)水平上的表達；通過克隆和測序排布出敏感菌K1及耐藥菌K3 orfX區(qū)的基因分布，與GenBank中的同源序列進行比較并分析其特征；SmaⅠ消化染色體DNA后，脈沖場電泳分離消化產(chǎn)物，Southern blot檢測orfX和mecA在染色體上的位置關(guān)系；分離、純化細菌膜蛋白后應用PBP assay鑒定出松鼠葡萄球菌的青霉素結(jié)合蛋白圖譜以及各個PBPs對β-內(nèi)酰胺類抗生素的親和力；分離、純化細胞壁，高壓液相法分析細胞壁成分。 結(jié)果：經(jīng)過抗生素選擇壓力，篩選出K3的homo*衍生菌K3HO，K3HW，K3HWO。K3對苯唑西林呈異質(zhì)性耐藥，K3 homo*衍生菌對苯唑西林高度均一耐藥，MIC＞400μg／ml。在抗生素存在的情況下，K3自發(fā)分化為K3w和K3y的現(xiàn)象被抑制，分化出的黃，白菌落均含有松鼠葡萄球菌mecA和金黃色葡萄球菌mecA。同時，金黃色葡萄球菌的mecA被大量誘導，但Northern blot檢測不到松鼠葡萄球菌mecA的表達。K3 homo*衍生菌的金黃色葡萄球菌mecA表達輕微提高，但也未能檢測到松鼠葡萄球菌mecA在RNA水平的表達。K3和K8分別為SCCmecⅢB和SCCmecⅢA型，SCCmec準確的插入到orfX區(qū)。K1和K3 orfX區(qū)的基因排布和表皮葡萄球菌和溶血葡萄球菌相似而和金黃色葡萄球菌差異較大。orfX和mecA基因在染色體上的位置隨菌株不同而不同。鑒定了6種PBPs，其中PBP4即為松鼠葡萄球菌mecA的表達產(chǎn)物，對苯唑西林呈低親和力。PBP6在苯唑西林耐藥性方面可能發(fā)揮了重要作用。苯唑西林耐藥菌細胞壁交聯(lián)率高于敏感菌，但交聯(lián)率的高低和菌株的MIC值并不呈線形關(guān)系。在抗生素壓力下，金黃色葡萄球菌的mecA產(chǎn)物能利用松鼠葡萄球菌細胞壁合成前體合成松鼠葡萄球菌的細胞壁。結(jié)論：在兩個mecA共存的松鼠葡萄球菌中，金黃色葡萄球菌mecA和抗藥性直接相關(guān)，但也不能排除松鼠葡萄球菌mecA的作用。orfX和mecA位于基因重組的熱點或附近，這有利于松鼠葡萄球菌獲得新的SCCmec或松鼠葡萄球菌mecA的轉(zhuǎn)移。松鼠葡萄球菌細胞壁的高度交聯(lián)率和耐藥性相關(guān)，，這不同于敏感和耐藥的金黃色葡萄球菌細胞壁特征。松鼠葡萄球菌的mecA和金黃色葡萄球菌mecA在細胞壁的合成中具有相互替代性。
[Abstract]:Staphylococcus aureus is the cause of nosocomial infection in one of the most important pathogens of infection and community-acquired. Beta lactam antibiotics have weapons is effective for the treatment of Staphylococcus aureus infection, but with methicillin resistant Staphylococcus aureus (Methicillin resistant Staphylococcus aureus, MRSA) gradually increase the separation rate that caused by Staphylococcus aureus infection become clinically difficult problem. Drug resistance genes were mainly determined binding protein 2A mecA.mecA encoding penicillin (penicillin-binding protein 2A, PBP2A) to very low affinity of beta lactam antibiotics, when the natural PBP beta lactam antibiotics when saturated, peptide function catalytic synthesis of cell wall.MecA gene of Staphylococcus aureus is not inherent gene PBP2A can replace these PBPs, but exogenous genes, their sources are very much that. Before that is obtained from the s.sciuri. Most s.sciuri mecA does not provide resistance, but the activation of s.sciuri mecA into sensitive Staphylococcus aureus can make the resistance increase greatly, the cell wall using Staphylococcus aureus mecA gene product s.sciuri the cell wall synthesis of the precursors of Staphylococcus aureus Staphylococcus K3. Recently, we have isolated with s.sciuri mecA containing mecA of Staphylococcus aureus, K3 yellow colonies, can spontaneously differentiate into K3y in the presence of no antibiotic (yellow colony) and K3w (white). The former. And genotype and their parental bacteria are exactly the same, the latter contains only s.sciuri mecA and lost mecA. of Staphylococcus aureus in this paper according to the two mecA gene in Staphylococcus sciuri Drug resistance and the role of cell wall synthesis are studied.
Objective: To study the features of biological characteristics and gene s.sciuri in order to further understand the drug resistance of Staphylococcus aureus mecA gene from MRSA strain as well as the resistance mechanism; at the same time, understanding the resistance mechanism of clinical isolates increasing s.sciuri. Methods: the antibiotic selective pressure from highly homogeneous resistant strains of squirrel grapes the application of E-test and K3; PAP test to detect the resistance of Staphylococcus to oxacillin; PCR amplification of two mecA genes and the preparation of the DNA probe; multiplex PCR for strain K3 and K8 SCCmec; Northern blot and Western blot were used to detect the expression of two mecA genes at the transcriptional level and protein level the cloning and sequencing; through the arrangement of the distribution of sensitive bacteria resistant bacteria K3 K1 and orfX region of the gene, compared with the homologous sequences in GenBank And analyzes its characteristics; Sma I digestion of chromosomal DNA, pulsed field gel electrophoresis separation of the digestion products, Southern blot position detection of orfX and mecA on the chromosome; separation, a s.sciuri penicillin binding protein profiles and the various PBPs of beta lactam antibiotics using PBP assay affinity purified bacterial membrane protein separation and purification; cell wall, cell wall composition analysis of high pressure liquid chromatography method.
Results: after antibiotic selection pressure, screened K3 homo* derived bacteria K3HO, K3HW, K3HWO.K3 showed heterogeneity of oxacillin resistant bacteria, K3 homo* derivatives of oxacillin resistant highly homogeneous, MIC > 400 g / ml. in the presence of antibiotics, K3 K3w and K3y for spontaneous differentiation phenomenon inhibition of the differentiation of the yellow, white colonies contain Staphylococcus mecA and Staphylococcus aureus mecA. and Staphylococcus aureus mecA was induced by Northern, but blot could not detect s.sciuri mecA expression.K3 homo* derived strains of Staphylococcus aureus mecA expression improved slightly, but also failed to detect to s.sciuri mecA in RNA the expression of.K3 and K8 were SCCmec B and SCCmec III type III A, SCCmec accurately inserted into the orfX region of.K1 and K3 orfX gene arrangement and Staphylococcus epidermidis and Staphylococcus haemolyticus and Staphylococcus and similar On the chromosomes of Staphylococcus aureus in different.OrfX and mecA genes with different strains and different location. 6 types of PBPs were identified, of which PBP4 is the expression product of s.sciuri mecA, to oxacillin showed low affinity of.PBP6 may play an important role in oxacillin resistance oxacillin resistant bacteria cells. Wall cross-linking rate is higher than the sensitive bacteria, but the crosslinking rate and the strains showed a linear relationship. The MIC value is not in the antibiotic pressure, cell wall using s.sciuri cell wall synthesis precursor for synthesis of s.sciuri mecA product of Staphylococcus aureus. Conclusion: in the two mecA coexistence of s.sciuri in mecA, Staphylococcus aureus and drug resistance is directly related to, but also can not rule out the role of.OrfX and mecA s.sciuri mecA or near the hot spot in gene recombination, which is conducive to grape ball squirrels The transfer of bacteria to get a new SCCmec or s.sciuri mecA. S.sciuri cell wall related high crosslinking rate and drug resistance, which is different from susceptible and resistant Staphylococcus aureus cell wall characteristics. Staphylococcus mecA and Staphylococcus aureus mecA replaced with each other in the synthesis of the cell wall.

【學位授予單位】：天津醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2007
【分類號】：R378.2

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