本文選題：腸桿菌 + 質(zhì)粒��； 參考：《華南農(nóng)業(yè)大學(xué)》2016年碩士論文

【摘要】：近年來,隨著多重耐藥革蘭陰性菌的增加,抗革蘭氏陰性菌感染藥物的選擇越來越有限。多粘菌素作為“最后一道防線”被重新用于臨床治療多重耐藥革蘭氏陰性菌引起的感染。細菌對多粘菌素類藥物的耐藥機制往往由染色體介導(dǎo),但本研究在國際上,首次發(fā)現(xiàn)了可水平轉(zhuǎn)移的質(zhì)粒介導(dǎo)的黏菌素耐藥機制MCR-1。以上海某一豬場分離的大腸桿菌為研究對象,隨機選取一株黏菌素的最小抑菌濃度為8 mg/L的菌株SHP45進行研究。采用PCR方法檢測引起多粘菌素耐藥的突變基因(pmr AB,pho PQ和mgr B),結(jié)果未檢測到與多粘菌素耐藥有關(guān)的基因突變。通過接合實驗成功將黏菌素耐藥性質(zhì)粒(p HNSHP45)轉(zhuǎn)移到受體菌中,接合頻率高達10-1~10-3,且p HNSHP45質(zhì)粒能通過接合或轉(zhuǎn)化轉(zhuǎn)入肺炎克雷伯菌中,并介導(dǎo)黏菌素耐藥。通過高通量測序獲得該質(zhì)粒全序列,發(fā)現(xiàn)該質(zhì)粒全長為62105bp,其G+C為43.0%,具有典型的Inc I2型質(zhì)粒骨架,并發(fā)現(xiàn)一個1626bp的開放閱讀框與插入序列ISAp I1相連,推測此為該質(zhì)粒從外源獲得的序列,該基因被命名為mcr-1。氨基酸序列分析結(jié)果顯示MCR-1與磷酸乙醇胺轉(zhuǎn)移酶有60%左右的相似性,細菌脂質(zhì)A的ESI-MS分析結(jié)果證實了MCR-1功能,即通過修飾細菌細胞膜上的脂質(zhì)A而介導(dǎo)對黏菌素耐藥。對原菌SHP45和接合子進行傳代實驗,采用S1-PFGE和雜交的方法檢測該質(zhì)粒的穩(wěn)定性,分別在含黏菌素和不含黏菌素的LB溶液傳代培養(yǎng)14代,發(fā)現(xiàn)質(zhì)粒p HNSHP45仍能夠穩(wěn)定地存在接合子和原菌SHP45中。采用PCR方法進一步檢測了mcr-1在動物源大腸桿菌中的傳播及擴散情況。PCR結(jié)果顯示,在2011~2014年生肉分離的523份大腸桿菌檢測到78株(15%)攜帶mcr-1,屠宰前動物分離的804份大腸桿菌檢測到166株(21%)攜帶mcr-1,表明mcr-1已在動物源腸桿菌中廣泛傳播,且陽性樣本的比例在逐年增加。本研究在國際上首次發(fā)現(xiàn)了質(zhì)粒介導(dǎo)的黏菌素耐藥基因mcr-1,mcr-1位于接合性Inc I2型質(zhì)粒上,與插入序列ISAp I1相連。MCR-1屬于磷酸乙醇胺轉(zhuǎn)移酶,通過修飾細菌細胞膜上脂質(zhì)A而介導(dǎo)對黏菌素的耐藥。mcr-1基因已在食品動物和動物性食品大腸桿菌中廣泛傳播,且該基因位于接合性質(zhì)粒上,極易傳播擴散,從分子機制上解釋了目前國內(nèi)多粘菌素耐藥性迅速升高的原因,為正確評估多粘菌素藥物在動物中的使用提供依據(jù)。
[Abstract]:In recent years, with the increase of multidrug resistant Gram-negative bacteria, the choice of anti-Gram-negative bacteria infection drugs is more and more limited. Polymyxin as a "last line of defense" has been reused to treat multidrug-resistant gram-negative infections. The mechanism of bacterial resistance to polymyxin is usually mediated by chromosomes, but in this study, the horizontal transferable plasmide-mediated mechanism of myxin resistance MCR-1 was first found in the world. Escherichia coli isolated from a pig farm in Shanghai was used as the research object. A strain SHP45 with the minimum inhibitory concentration of myxin 8 mg / L was selected at random. The mutated genes (pmr ABPO PQ and mgr B) causing polymyxin resistance were detected by PCR. No mutation related to polymyxin resistance was detected. The plasmids pHNSHP45 were successfully transferred to the recipient bacteria by conjugation experiment. The binding frequency of pHNSHP45 plasmid was as high as 10 ~ (-1) ~ 10 ~ (-3), and pHNSHP45 plasmid could be transfered into Klebsiella pneumoniae by conjugation or transformation, and it could mediate the drug resistance of myxin to Klebsiella pneumoniae. The whole sequence of the plasmid was obtained by high-throughput sequencing. It was found that the full length of the plasmid was 62105bpand its G C was 43.0. it had the typical framework of ISAI2 plasmid, and found that an open reading frame of 1626bp was linked to the inserted sequence ISApI1. The gene was named mcr-1. Amino acid sequence analysis showed that MCR-1 was similar to ethanolamine phosphotransferase by about 60%. ESI-MS analysis of bacterial lipid A confirmed the function of MCR-1, that is, MCR-1 mediated resistance to myxin by modifying lipid A on bacterial cell membrane. The stability of the plasmid was detected by S1-PFGE and hybridization. The plasmids were subcultured in LB solution containing myxine and no mucin for 14 generations, respectively. It was found that the plasmids pHNSHP45 still exist stably in the conjugates and protobacteria SHP45. The spread and diffusion of mcr-1 in Escherichia coli of animal origin were further detected by PCR method. From 2011 to 2014, 78 strains (15%) of Escherichia coli isolated from raw meat and 804 strains (21%) of Escherichia coli from animals before slaughter were detected to carry mcr-1, indicating that mcr-1 has been widely spread in Enterobacter faecalis and the proportion of positive samples has been increasing year by year. In this study, the plasmide-mediated myxin resistance gene mcr-1mcr-1 was first found to be located on the plasmids of conjugated Inc I2 type, which was linked to the inserted sequence ISApI1. MCR-1 belongs to ethanolamine phosphate transferase. The drug resistance. Mcr-1 gene mediated by the modification of lipid A on the cell membrane of bacteria has been widely spread in food animals and animal food Escherichia coli, and the gene is located on the conjugate particles and is easy to spread. The molecular mechanism explains the rapid increase of drug resistance of polymyxin in China, which provides a basis for the correct evaluation of the use of polymyxin in animals.
【學(xué)位授予單位】：華南農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R978

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1 陳冠容;;多粘菌素臨床應(yīng)用進展及應(yīng)對超級細菌[J];醫(yī)藥導(dǎo)報;2011年02期

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本文編號：2061642