松鼠葡萄球菌mecA和金黄色葡萄球菌mecA在松鼠葡萄球菌耐药机制及细胞壁合成中的作用

发布时间：2018-04-16 00:15

  本文选题：金黄色葡萄球菌 + 松鼠葡萄球菌　； 参考：《天津医科大学》2007年博士论文

【摘要】： 金黄色葡萄球菌是引起医院内获得性感染和社区获得性感染的重要致病菌之一。β-内酰胺类抗生素曾经是治疗金黄色葡萄球菌感染的有效武器，但随着耐甲氧西林的金黄色葡萄球菌(Methicillin resistant Staphylococcus aureus，MRSA)分离率的逐渐增加，金黄色葡萄球菌引起的感染成为临床上非常棘手的问题。耐药的主要决定基因为mecA。mecA编码的青霉素结合蛋白2A(penicillin-binding protein 2A，PBP2A)对β-内酰胺类抗生素亲和力很低，当固有的PBP被β-内酰胺类抗生素饱和时，PBP2A可替代这些PBPs的转肽功能催化细胞壁合成。mecA基因并非金黄色葡萄球菌的固有基因，而是一外源基因，其来源有很多假设，目前认为是从松鼠葡萄球菌中获得。大多数松鼠葡萄球菌的mecA并不提供抗药性，但把活化的松鼠葡萄球菌mecA引入到敏感的金黄色葡萄球菌可使后者的抗药性大大增加，松鼠葡萄球菌的mecA基因产物能够利用金黄色葡萄球菌的细胞壁前体合成金黄色葡萄球菌的细胞壁。最近，我们分离到了既含有松鼠葡萄球菌的mecA又含有金黄色葡萄球菌的mecA的松鼠葡萄球菌K3，K3为黄色菌落，在没有抗生素存在时能自发分化为K3y(黄色菌落)和K3w(白色菌落)。前者表型和基因型与亲代菌完全相同，后者仅含有松鼠葡萄球菌mecA而丢失了金黄色葡萄球菌的mecA。本论文将针对这两个mecA基因在松鼠葡萄球菌的耐药性及细胞壁合成中的作用进行研究。 目的：进一步研究松鼠葡萄球菌的生物学特征和基因特征以期更深入的理解金黄色葡萄球菌耐药性基因mecA来源以及MRSA菌株的耐药机制；同时，认识临床上分离率日益增加的松鼠葡萄球菌的耐药机制。方法：通过抗生素压力选择出松鼠葡萄球菌K3的高度均一耐药株；应用E-test和PAP试验来检测松鼠葡萄球菌对苯唑西林的耐药性；PCR扩增两个mecA基因并制备DNA探针；多重PCR用于菌株K3和K8的SCCmec分型；应用Northern blot和Western blot分别检测两个mecA基因在转录水平和蛋白质水平上的表达；通过克隆和测序排布出敏感菌K1及耐药菌K3 orfX区的基因分布，与GenBank中的同源序列进行比较并分析其特征；SmaⅠ消化染色体DNA后，脉冲场电泳分离消化产物，Southern blot检测orfX和mecA在染色体上的位置关系；分离、纯化细菌膜蛋白后应用PBP assay鉴定出松鼠葡萄球菌的青霉素结合蛋白图谱以及各个PBPs对β-内酰胺类抗生素的亲和力；分离、纯化细胞壁，高压液相法分析细胞壁成分。 结果：经过抗生素选择压力，筛选出K3的homo*衍生菌K3HO，K3HW，K3HWO。K3对苯唑西林呈异质性耐药，K3 homo*衍生菌对苯唑西林高度均一耐药，MIC＞400μg／ml。在抗生素存在的情况下，K3自发分化为K3w和K3y的现象被抑制，分化出的黄，白菌落均含有松鼠葡萄球菌mecA和金黄色葡萄球菌mecA。同时，金黄色葡萄球菌的mecA被大量诱导，但Northern blot检测不到松鼠葡萄球菌mecA的表达。K3 homo*衍生菌的金黄色葡萄球菌mecA表达轻微提高，但也未能检测到松鼠葡萄球菌mecA在RNA水平的表达。K3和K8分别为SCCmecⅢB和SCCmecⅢA型，SCCmec准确的插入到orfX区。K1和K3 orfX区的基因排布和表皮葡萄球菌和溶血葡萄球菌相似而和金黄色葡萄球菌差异较大。orfX和mecA基因在染色体上的位置随菌株不同而不同。鉴定了6种PBPs，其中PBP4即为松鼠葡萄球菌mecA的表达产物，对苯唑西林呈低亲和力。PBP6在苯唑西林耐药性方面可能发挥了重要作用。苯唑西林耐药菌细胞壁交联率高于敏感菌，但交联率的高低和菌株的MIC值并不呈线形关系。在抗生素压力下，金黄色葡萄球菌的mecA产物能利用松鼠葡萄球菌细胞壁合成前体合成松鼠葡萄球菌的细胞壁。结论：在两个mecA共存的松鼠葡萄球菌中，金黄色葡萄球菌mecA和抗药性直接相关，但也不能排除松鼠葡萄球菌mecA的作用。orfX和mecA位于基因重组的热点或附近，这有利于松鼠葡萄球菌获得新的SCCmec或松鼠葡萄球菌mecA的转移。松鼠葡萄球菌细胞壁的高度交联率和耐药性相关，，这不同于敏感和耐药的金黄色葡萄球菌细胞壁特征。松鼠葡萄球菌的mecA和金黄色葡萄球菌mecA在细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.

【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R378.2

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