猪不同生长阶段耐黏菌素大肠埃希菌的动态变化研究

发布时间：2018-04-01 01:31

本文选题：猪　切入点：黏菌素　出处：《吉林农业大学》2017年硕士论文

【摘要】：多黏菌素在20世纪60年代被广泛用于治疗革兰氏阴性菌感染,因多黏菌素类药物抗菌谱窄且具有较严重的肾毒性和神经毒性等不良反应,在20世纪80年代,被抗菌谱更广、不良反应更小的抗菌药物如β-内酰胺类抗生素所替代,在临床上逐渐被限制使用,但是近几年随着耐碳青霉烯类抗生素菌株的出现,多黏菌素类抗生素作为治疗多重耐药菌感染,又被重新应用于临床,使用频率也趋增加,并被视为临床抗感染的最后选择。2015年刘建华首次提出了肠杆菌科质粒介导的mcr-1耐黏菌素机制,相关研究证实mcr-1基因不仅可以从动物身上分离得到,从人和肉类食物中也可分离得到,且分离出的耐黏菌素的菌株主要为大肠埃希菌。为比较分析获得我国不同地区猪不同生长阶段粪便中携带耐黏菌素大肠埃希菌的本地数据,阐明黏菌素耐药基因扩散和水平传播机制,本研究采集来自吉林省长春市、吉林省梅河口市、甘肃省张掖市、甘肃省庆阳市四个地区共563份猪粪便样品,通过黏菌素抗性选择性培养基培养、PCR方法鉴定耐黏菌素大肠埃希菌;使用PCR的方法筛选黏菌素耐药基因;使用多重PCR的方法对耐黏菌素大肠埃希菌进行系统进化分群、质粒分型实验;通过最小抑菌浓度(MIC)实验确定耐黏菌素大肠埃希菌分离株对黏菌素的耐药值;通过接合转移实验来确定耐药基因扩散、水平传播机制。结果显示:从来自吉林省长春市、吉林省梅河口市、甘肃省张掖市、甘肃省庆阳市四个地区共563份猪粪便样品分离鉴定出273株耐黏菌素大肠埃希菌,总分离率为48.5%。针对273株分离株检测黏菌素耐药基因mcr-1,mcr-1耐药基因的检出率为100%。对273株耐黏菌素大肠埃希菌分离株进行了系统进化分群,结果显示除庆阳市育肥阶段外,长春市、梅河口市和张掖市三个地区在育成、育肥阶段均以A群为主要优势群,B1群次之。四个不同地区分离株在哺乳阶段的系统进化分群结果存在稍微差别。对273株耐黏菌素大肠埃希菌分离株进行质粒复制子基因分型。大肠埃希菌常见质粒复制子基因分型检测,结果显示共检测到11种质粒型,不同地区的分离株均以IncFIB型质粒(41.8%,114/273)为主,其次为IncY型质粒(14.3%,39/273),IncFIC型质粒(8.8%,24/273)等;可携带mcr-1基因质粒复制子基因分型检测,结果显示不同地区的分离株主要为X4型质粒(38.1%,104/273),其次为I2型质粒(26.4%,72/273)等。挑选四个不同地区不同生长阶段的172株样品分离株进行接合转移实验,结果发现有123株接合转移成功,接合转移发生率为71.5%(123/172),且接合子均携带mcr-1基因,说明mcr-1基因位于接合性质粒上,并且可以伴随着质粒发生转移、传播和扩散。针对挑选的四个不同地区不同生长阶段的172株样品分离株及接合转移成功获得的123株接合子做了最小抑菌浓度(MIC)实验,结果显示不同地区分离株对黏菌素的MIC值主要为8μg/mL(83.7%144/172),其次为4μg/mL(14.5%25/172)、16μg/mL(1.8%3/172)。在发生水平转移后大部分接合子对黏菌素的MIC值保持一致(86.2%106/123),部分接合子发生升高(7.3%9/123)或降低(6.5%8/123)的变化。本研究通过对不同地区猪不同生长阶段耐黏菌素大肠埃希菌的监测所获得的数据,初步掌握了猪不同生长阶段耐黏菌素大肠埃希菌的动态变化,为mcr-1耐黏菌素机制的研究提供参考数据。
[Abstract]:Polymyxin in 1960s are widely used in the treatment of gram negative bacterial infections, due to adverse reactions, polymyxin drugs narrow antimicrobial spectrum and has a more severe renal toxicity and neurotoxicity in 1980s, by the broader antibacterial spectrum, less adverse drug reaction such as beta lactam antibiotics substitute in clinic has been restricted but in recent years with the emergence of carbapenem resistant strains, polymyxin antibiotics for the treatment of multi drug resistant bacteria infection, and was re used in the clinical use of frequency is also increasing, and is considered the final choice.2015 for clinical anti infection Liu Jianhua first proposed colistins mechanism mcr-1 resistant Enterobacteriaceae plasmid mediated, related studies have confirmed that mcr-1 gene not only can be isolated from the animal body, from human and meat food can also be separated, and the separated resistance Colistin was mainly Escherichia coli. Comparison and analysis of the local data carrying resistance to polymyxin E. coli for pigs in different regions of China at different growth stages in feces, clarify the colistin resistant gene diffusion and horizontal transmission mechanism, this study collected from Jilin City, Jilin province governor spring, Meihekou City, Gansu City, Zhangye Province Qingyang City, Gansu Province four area, a total of 563 swine fecal samples by selective culture medium of colistin resistant, resistant to polymyxin PCR method for identification of Escherichia coli; using the PCR method for screening of colistin resistance gene; the phylogenetic clustering of colistin resistant Escherichia coli by multiplex PCR method, plasmids experiment; the minimum inhibitory concentration (MIC) experiment to determine the resistance to colistin Escherichia coli isolates of colistin resistance value; by conjugation experiment to determine the spread of resistance genes, the level of transfer Sowing mechanism. The results showed that the Changchun from Jilin Province, Jilin city of Meihekou Province, Gansu Province, Zhangye City, Gansu Province, Qingyang City, a total of four area 563 swine fecal samples were isolated and identified 273 strains of Escherichia coli in myxomycetes, the total isolation rate is 48.5%. for 273 isolates detected colistin resistant gene mcr-1 mcr-1, the detection rate of resistant gene 100%. in Escherichia coli isolated from the phylogenetic grouping of 273 strains were resistant to slime, the result shows that in the city of Qingyang City, fattening stage, Changchun, Meihekou city and Zhangye city in three areas of incubation, the fattening stage in A group was the dominant group, B1 group the phylogenetic clustering results. Isolates in lactation stage are slightly different in different regions. Four of 273 strains of colistin resistant Escherichia coli isolates were plasmid replicon. The genotype of Escherichia coli plasmid replicon common genotype. Test results show that the 11 type plasmids were detected in different regions of the isolates were IncFIB type plasmid (41.8%, 114/273), followed by type IncY plasmid (14.3%, 39/273), IncFIC (8.8%, 24/273) plasmid; mcr-1 gene carrying plasmid replicon genotyping results showed. Different regions of the isolates were mainly X4 plasmid (38.1%, 104/273), followed by type I2 plasmid (26.4%, 72/273). The selection of four different regions in different growth stages of 172 strains isolates by conjugation experiment, the results showed that 123 strains conjugation, conjugation rate was 71.5% (123/172), and the zygote were carrying mcr-1 gene, mcr-1 gene located on the plasmid, and may be accompanied by a plasmid transfer and spread. In four different regions selected in different growth stages of 172 strains isolated from samples and conjugative transfer success 123 strains of zygote had minimal inhibitory concentration (MIC) experiments, the results showed that different isolates of colistin MIC value of 8 g/mL (83.7%144/172), followed by 4 g/mL (14.5%25/172), 16 g/mL (1.8%3/172). In the event of horizontal transfer most of zygote to colistin MIC the value of consistency (86.2%106/123), merozygote were significantly increased (7.3%9/123) or lower (6.5%8/123) changes. This study through the monitoring of different regions in different growth stages of pig resistance to colistin of Escherichia coli in data obtained from the initial grasp of the dynamic changes of pigs in different growth stages of colistin resistant Escherichia coli, provide the reference data of colistin resistant mechanism of mcr-1.

【学位授予单位】：吉林农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.6

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