奶牛乳腺炎来源的葡萄球菌甲氧西林抗性传播的机制研究

发布时间：2018-01-09 02:03

  本文关键词：奶牛乳腺炎来源的葡萄球菌甲氧西林抗性传播的机制研究　出处：《西北农林科技大学》2017年博士论文　论文类型：学位论文

  更多相关文章： 葡萄球菌 甲氧西林 抗性表型 SCCmec 基因转移

【摘要】：葡萄球菌是导致人畜共患病的主要病原菌之一,在畜禽上可引起急性或慢性传染病,给畜禽带来严重的健康威胁,给畜禽养殖业造成极大的经济损失。近年来抗生素的过度使用催生了耐甲氧西林葡萄球菌(MRS),这类葡萄球菌外源性获得了一种染色体盒式结构(SCCmec)。位于SCCmec上的mecA(或者mecC)基因编码的PBP2a蛋白参与细胞壁的合成,对内酰胺类抗生素有很低的亲和力,从而介导MRS对内酰胺类药物的抗性。临床上,MRS的检测通常是通过抗性表型筛选的方法来实现的。大量间接证据表明MRS菌株之所以大量出现并广泛传播,是因为SCCmec可以在不同菌种和不同菌株之间进行水平基因转移。水平基因转移是抗性传播的主要方式,SCCmec从基因组上切除是SCCmec转移到其他菌株的起始步骤,也是最为关键的一步。SCCmec的切除是由自身携带的编码盒式染色体重组酶基因ccr介导的。目前为止,常用抗生素如何调控SCCmec切除和转移的信号通路并不清楚,亟待研究。近年来,一种新型的MRS菌株,即表型敏感,mecA阳性的耐甲氧西林葡萄球菌(OS-MRS)不断被报道。这类葡萄球菌含有SCCmec/mecA,却没有抗性表型,会被错误的诊断为甲氧西林敏感葡萄球菌。而OS-MRS菌株在内酰胺类抗生素的刺激下,可以在短时间内由敏感表型转变为高抗表型,苯唑西林最低抑菌浓度高达128μg/mL以上。这会导致治疗失败,病情加重等情况,对人类和畜禽健康,甚至生命造成巨大威胁。因此,找到参与OS-MRS菌株表型调控和表型转变的关键性调节因子就成了解决这类问题的首要任务,值得深入研究。实验一我们通过抗生素标准检测方法,对采集来的244个牛生鲜乳样品中是否残留抗生素进行了检测,结果显示,超过50%的样品含有抗生素。随机选择了39个含有抗生素的样品,进一步检测了其中5种特定抗生素(环丙沙星、氯霉素、磺胺嘧啶、链霉素和四环素)的具体残留量。结果发现,在选取的39个含抗生素样品中,环丙沙星的残留量均超过欧盟制定的最大残留限制标准(MRLs);此外,2种、3种、4种和5种抗生素在单样品中同时超出标准(MRLs)的样品数量分别是11个、9个、13个和2个。试验二我们通过多种分子生物学手段,试图确定畜牧中广泛使用的一些抗生素是通过何种信号通路,最终促进重组酶ccr基因的表达和SCCmec的转移起始。从试验结果中发现,很多抗生素能显著促进重组酶ccr基因的表达和SCCmec的切除,包括内酰胺类,糖肽类,四环素类,磺胺类,喹诺酮类等抗生素。在这些抗生素中,靶向DNA的抗生素能在浓度很低的情况下诱导ccr的表达。因此,我们推测,抗生素可能通过激活SOS修复途径,进而上调ccr的表达。经过体内的敲除实验和体外的凝胶阻滞实验验证,我们发现,抗生素在一定剂量下能够引起葡萄球菌的DNA损伤,DNA的损伤激活SOS信号通路,信号通路中的重要蛋白LexA从ccr基因的启动子上脱落下来,Ccr的表达促进SCCmec的切除。试验三我们研究了耐甲氧西林葡萄球菌中甲氧西林抗性表型的调控机制。使用牛奶源分离得到的葡萄球菌菌株作为实验材料,通过基因表达水平差异比较,发现调节性因子BlaR1和BlaI的表达量是影响抗性表型和抗性转变的关键因素。在OS-MRS菌株中,BlaR1和BlaI都是高表达,在无抗生素诱导的情况下,高表达的BlaI能抑制mec A的表达,从而降低抗性表型;而在抗生素诱导的情况下,高表达的BlaR1可以通过活化解除BlaI的抑制作用,促进PBP2a在短时间内高表达,以恢复抗性表型应对抗生素的刺激。综上所述,实验一的结果建议,牛生鲜乳中抗生素残留的常规检测对于避免畜牧生产中抗生素的不合理使用是必要的。试验二的结果详细阐明了多种抗菌制剂促进葡萄球菌甲氧西林抗性传播的机制,意义重大。在畜禽养殖上,甚至在人类疾病的临床治疗上,这对我们如何设计治疗方案和所用抗生素剂量以应对MRS感染产生直接影响。同时,为抗菌制剂的研发提供了新的方向,即如何以一种有效的但不会促进抗生素耐药性扩散的思维方式去进行新药研发。实验三的结果提示,在对MRS,包括OS-MRS进行筛选鉴定的过程中,要把bla基因簇的影响考虑进去。另外,研发靶向失活BlaR1的抑制剂是解决治疗OS-MRS感染困难的有效途径之一。
[Abstract]:Staphylococcus aureus is one of the leading pathogen of zoonosis bacteria in livestock and poultry can cause acute or chronic infectious disease, bring serious health threat to livestock, causing great economic losses to the livestock industry. In recent years, the excessive use of antibiotics has spawned methicillin-resistant Staphylococcus aureus (MRS), this type of Staphylococcus aureus exogenous acquired a chromosome cassette structure (SCCmec). On the SCCmec mecA (or mecC) gene encoding the PBP2a protein synthesis in the cell wall, has a very low affinity for - lactam antibiotics, which mediates resistance of MRS lactam drugs. Clinically, MRS detection is usually through the method of resistance phenotype screening to achieve. A large number of indirect evidence that the MRS strain has appeared in large numbers and widely spread, because SCCmec can be between different species and different strains of horizontal gene transfer levels. Gene transfer is the main way to the spread of resistance, SCCmec from genome SCCmec resection is transferred to the initial step of other strains, but also the most crucial step is.SCCmec resection cassette chromosome recombinase gene encoding CCR mediated by their own guide. Now, how to regulate the signal pathway of antibiotics SCCmec resection and metastasis is not clear, need to study. In recent years, a new type of MRS strains, namely sensitive phenotype, methicillin-resistant Staphylococcus aureus positive mecA (OS-MRS) has been reported. This kind of Staphylococcus aureus containing SCCmec/mecA, but there is no resistance phenotype, will be mistaken as methicillin sensitive Staphylococcus and stimulating strain OS-MRS, amide antibiotics, can in a short time by the sensitive phenotype changed into high resistance phenotype, oxacillin mic up to more than 128 g/mL. This will lead to treatment failure and disease Exacerbations etc., on human and animal health, and even cause a huge threat to life. Therefore, to find the key to participate in phenotypic regulation and phenotype transformation of OS-MRS regulator has become the primary task to solve this kind of problems, it is worthy of further study. Our experiment through antibiotic standard detection methods of antibiotic residues is 244 bovine fresh milk samples were collected for detection was carried out, results showed that more than 50% of the samples containing antibiotics. Randomly selected 39 samples containing antibiotics, further detection of these 5 specific antibiotics (ciprofloxacin, chloramphenicol, sulfadiazine, streptomycin and tetracycline) specific residues. The results showed that in 39 A sample containing antibiotic selection, maximum residue limits of residues of ciprofloxacin were more than the European Union (MRLs); in addition, 2, 3, 4 and 5 kinds of antibiotics in a single sample at the same time Beyond the standard (MRLs) of the number of samples were 11, 9, 13 and 2. Two we test through a variety of molecular biology methods, trying to determine some antibiotics widely used in animal husbandry through which signal pathway, ultimately promote the transfer of initial SCCmec and the expression of CCR recombinase gene. According to experimental results in a lot of antibiotics can significantly promote the removal of SCCmec and the expression of CCR recombinase gene, including lactams, glycopeptides, tetracyclines, sulfonamides, quinolones and other antibiotics. These antibiotics, targeting DNA antibiotics can induce the expression of CCR in very low concentration conditions. Therefore, we speculate that antibiotics may activate SOS repair pathways, and the expression of CCR. After the gel retardation experiments in vitro and in vivo experiments in knock we found that antibiotics could be caused by Staphylococcus aureus DN in certain dose A damage, DNA damage activated SOS signaling pathway, an important protein in the LexA signaling pathway falling down from the promoter of the CCR gene, SCCmec was promoting the expression of Ccr. Experiment three we study the regulation mechanism of methicillin-resistant Staphylococcus aureus in methicillin resistant phenotype. The milk source separation of Staphylococcus aureus strain is obtained as the experimental material, the gene expression level difference, found the expression of regulatory factors BlaR1 and BlaI are key factors that influence the change of resistance phenotype and resistance. In the OS-MRS strains, BlaR1 and BlaI are highly expressed in the absence of antibiotic induced conditions, high expression of BlaI can inhibit MEC A, so as to reduce the resistance phenotype; and in the case of antibiotic induced, high expression of BlaR1 can inhibit the activation of BlaI release, promote the high expression of PBP2a in a short period of time, to restore the resistance phenotype to The stimulation of antibiotics. In summary, the results of the experiment suggest that routine detection of antibiotic residues in raw milk cow is necessary to avoid the irrational use of antibiotics in animal husbandry. The two test results elucidated the variety of antimicrobial agents to promote the mechanism of Staphylococcus aureus methicillin resistance dissemination is of great significance. In livestock and poultry breeding. Even in the clinical treatment of human diseases, the treatment scheme and how we design the antibiotic dose directly affect infection in response to MRS. At the same time, provides a new direction for the development of antimicrobial agents, namely how to promote an effective but not antibiotic resistance diffusion approaches to drug development. Three experiments suggest that in MRS, including the OS-MRS process of screening and identification, the effect of the BLA gene cluster into account. In addition, research targeted inactivation of BlaR1 Inhibitors are one of the effective ways to solve the difficulties in treating OS-MRS infection.

【学位授予单位】：西北农林科技大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S858.23
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本文编号：1399530