质粒介导的黏菌素耐药基因mcr-1的发现

发布时间：2018-06-24 13:14

本文选题：肠杆菌 + 质粒　；参考：《华南农业大学》2016年硕士论文

【摘要】：近年来,随着多重耐药革兰阴性菌的增加,抗革兰氏阴性菌感染药物的选择越来越有限。多粘菌素作为“最后一道防线”被重新用于临床治疗多重耐药革兰氏阴性菌引起的感染。细菌对多粘菌素类药物的耐药机制往往由染色体介导,但本研究在国际上,首次发现了可水平转移的质粒介导的黏菌素耐药机制MCR-1。以上海某一猪场分离的大肠杆菌为研究对象,随机选取一株黏菌素的最小抑菌浓度为8 mg/L的菌株SHP45进行研究。采用PCR方法检测引起多粘菌素耐药的突变基因(pmr AB,pho PQ和mgr B),结果未检测到与多粘菌素耐药有关的基因突变。通过接合实验成功将黏菌素耐药性质粒(p HNSHP45)转移到受体菌中,接合频率高达10-1~10-3,且p HNSHP45质粒能通过接合或转化转入肺炎克雷伯菌中,并介导黏菌素耐药。通过高通量测序获得该质粒全序列,发现该质粒全长为62105bp,其G+C为43.0%,具有典型的Inc I2型质粒骨架,并发现一个1626bp的开放阅读框与插入序列ISAp I1相连,推测此为该质粒从外源获得的序列,该基因被命名为mcr-1。氨基酸序列分析结果显示MCR-1与磷酸乙醇胺转移酶有60%左右的相似性,细菌脂质A的ESI-MS分析结果证实了MCR-1功能,即通过修饰细菌细胞膜上的脂质A而介导对黏菌素耐药。对原菌SHP45和接合子进行传代实验,采用S1-PFGE和杂交的方法检测该质粒的稳定性,分别在含黏菌素和不含黏菌素的LB溶液传代培养14代,发现质粒p HNSHP45仍能够稳定地存在接合子和原菌SHP45中。采用PCR方法进一步检测了mcr-1在动物源大肠杆菌中的传播及扩散情况。PCR结果显示,在2011~2014年生肉分离的523份大肠杆菌检测到78株(15%)携带mcr-1,屠宰前动物分离的804份大肠杆菌检测到166株(21%)携带mcr-1,表明mcr-1已在动物源肠杆菌中广泛传播,且阳性样本的比例在逐年增加。本研究在国际上首次发现了质粒介导的黏菌素耐药基因mcr-1,mcr-1位于接合性Inc I2型质粒上,与插入序列ISAp I1相连。MCR-1属于磷酸乙醇胺转移酶,通过修饰细菌细胞膜上脂质A而介导对黏菌素的耐药。mcr-1基因已在食品动物和动物性食品大肠杆菌中广泛传播,且该基因位于接合性质粒上,极易传播扩散,从分子机制上解释了目前国内多粘菌素耐药性迅速升高的原因,为正确评估多粘菌素药物在动物中的使用提供依据。
[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.
【学位授予单位】：华南农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R978

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