發(fā)布時(shí)間：2018-01-09 02:03

  本文關(guān)鍵詞：奶牛乳腺炎來源的葡萄球菌甲氧西林抗性傳播的機(jī)制研究　出處：《西北農(nóng)林科技大學(xué)》2017年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 葡萄球菌 甲氧西林 抗性表型 SCCmec 基因轉(zhuǎn)移

【摘要】：葡萄球菌是導(dǎo)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傳染病,給畜禽帶來嚴(yán)重的健康威脅,給畜禽養(yǎng)殖業(yè)造成極大的經(jīng)濟(jì)損失。近年來抗生素的過度使用催生了耐甲氧西林葡萄球菌(MRS),這類葡萄球菌外源性獲得了一種染色體盒式結(jié)構(gòu)(SCCmec)。位于SCCmec上的mecA(或者mecC)基因編碼的PBP2a蛋白參與細(xì)胞壁的合成,對(duì)內(nèi)酰胺類抗生素有很低的親和力,從而介導(dǎo)MRS對(duì)內(nèi)酰胺類藥物的抗性。臨床上,MRS的檢測通常是通過抗性表型篩選的方法來實(shí)現(xiàn)的。大量間接證據(jù)表明MRS菌株之所以大量出現(xiàn)并廣泛傳播,是因?yàn)镾CCmec可以在不同菌種和不同菌株之間進(jìn)行水平基因轉(zhuǎn)移。水平基因轉(zhuǎn)移是抗性傳播的主要方式,SCCmec從基因組上切除是SCCmec轉(zhuǎn)移到其他菌株的起始步驟,也是最為關(guān)鍵的一步。SCCmec的切除是由自身攜帶的編碼盒式染色體重組酶基因ccr介導(dǎo)的。目前為止,常用抗生素如何調(diào)控SCCmec切除和轉(zhuǎn)移的信號(hào)通路并不清楚,亟待研究。近年來,一種新型的MRS菌株,即表型敏感,mecA陽性的耐甲氧西林葡萄球菌(OS-MRS)不斷被報(bào)道。這類葡萄球菌含有SCCmec/mecA,卻沒有抗性表型,會(huì)被錯(cuò)誤的診斷為甲氧西林敏感葡萄球菌。而OS-MRS菌株在內(nèi)酰胺類抗生素的刺激下,可以在短時(shí)間內(nèi)由敏感表型轉(zhuǎn)變?yōu)楦呖贡硇?苯唑西林最低抑菌濃度高達(dá)128μg/mL以上。這會(huì)導(dǎo)致治療失敗,病情加重等情況,對(duì)人類和畜禽健康,甚至生命造成巨大威脅。因此,找到參與OS-MRS菌株表型調(diào)控和表型轉(zhuǎn)變的關(guān)鍵性調(diào)節(jié)因子就成了解決這類問題的首要任務(wù),值得深入研究。實(shí)驗(yàn)一我們通過抗生素標(biāo)準(zhǔn)檢測方法,對(duì)采集來的244個(gè)牛生鮮乳樣品中是否殘留抗生素進(jìn)行了檢測,結(jié)果顯示,超過50%的樣品含有抗生素。隨機(jī)選擇了39個(gè)含有抗生素的樣品,進(jìn)一步檢測了其中5種特定抗生素(環(huán)丙沙星、氯霉素、磺胺嘧啶、鏈霉素和四環(huán)素)的具體殘留量。結(jié)果發(fā)現(xiàn),在選取的39個(gè)含抗生素樣品中,環(huán)丙沙星的殘留量均超過歐盟制定的最大殘留限制標(biāo)準(zhǔn)(MRLs);此外,2種、3種、4種和5種抗生素在單樣品中同時(shí)超出標(biāo)準(zhǔn)(MRLs)的樣品數(shù)量分別是11個(gè)、9個(gè)、13個(gè)和2個(gè)。試驗(yàn)二我們通過多種分子生物學(xué)手段,試圖確定畜牧中廣泛使用的一些抗生素是通過何種信號(hào)通路,最終促進(jìn)重組酶ccr基因的表達(dá)和SCCmec的轉(zhuǎn)移起始。從試驗(yàn)結(jié)果中發(fā)現(xiàn),很多抗生素能顯著促進(jìn)重組酶ccr基因的表達(dá)和SCCmec的切除,包括內(nèi)酰胺類,糖肽類,四環(huán)素類,磺胺類,喹諾酮類等抗生素。在這些抗生素中,靶向DNA的抗生素能在濃度很低的情況下誘導(dǎo)ccr的表達(dá)。因此,我們推測,抗生素可能通過激活SOS修復(fù)途徑,進(jìn)而上調(diào)ccr的表達(dá)。經(jīng)過體內(nèi)的敲除實(shí)驗(yàn)和體外的凝膠阻滯實(shí)驗(yàn)驗(yàn)證,我們發(fā)現(xiàn),抗生素在一定劑量下能夠引起葡萄球菌的DNA損傷,DNA的損傷激活SOS信號(hào)通路,信號(hào)通路中的重要蛋白LexA從ccr基因的啟動(dòng)子上脫落下來,Ccr的表達(dá)促進(jìn)SCCmec的切除。試驗(yàn)三我們研究了耐甲氧西林葡萄球菌中甲氧西林抗性表型的調(diào)控機(jī)制。使用牛奶源分離得到的葡萄球菌菌株作為實(shí)驗(yàn)材料,通過基因表達(dá)水平差異比較,發(fā)現(xiàn)調(diào)節(jié)性因子BlaR1和BlaI的表達(dá)量是影響抗性表型和抗性轉(zhuǎn)變的關(guān)鍵因素。在OS-MRS菌株中,BlaR1和BlaI都是高表達(dá),在無抗生素誘導(dǎo)的情況下,高表達(dá)的BlaI能抑制mec A的表達(dá),從而降低抗性表型;而在抗生素誘導(dǎo)的情況下,高表達(dá)的BlaR1可以通過活化解除BlaI的抑制作用,促進(jìn)PBP2a在短時(shí)間內(nèi)高表達(dá),以恢復(fù)抗性表型應(yīng)對(duì)抗生素的刺激。綜上所述,實(shí)驗(yàn)一的結(jié)果建議,牛生鮮乳中抗生素殘留的常規(guī)檢測對(duì)于避免畜牧生產(chǎn)中抗生素的不合理使用是必要的。試驗(yàn)二的結(jié)果詳細(xì)闡明了多種抗菌制劑促進(jìn)葡萄球菌甲氧西林抗性傳播的機(jī)制,意義重大。在畜禽養(yǎng)殖上,甚至在人類疾病的臨床治療上,這對(duì)我們?nèi)绾卧O(shè)計(jì)治療方案和所用抗生素劑量以應(yīng)對(duì)MRS感染產(chǎn)生直接影響。同時(shí),為抗菌制劑的研發(fā)提供了新的方向,即如何以一種有效的但不會(huì)促進(jìn)抗生素耐藥性擴(kuò)散的思維方式去進(jìn)行新藥研發(fā)。實(shí)驗(yàn)三的結(jié)果提示,在對(duì)MRS,包括OS-MRS進(jìn)行篩選鑒定的過程中,要把bla基因簇的影響考慮進(jìn)去。另外,研發(fā)靶向失活BlaR1的抑制劑是解決治療OS-MRS感染困難的有效途徑之一。
[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.

【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：S858.23
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本文編號(hào)：1399530