不同来源肠球菌的耐药性调查及对氟苯尼考耐药机制的研究

发布时间：2018-09-13 15:53

【摘要】：肠球菌多药耐药的特点,以及耐万古霉素肠球菌的出现,已引起医学和兽医临床的广泛关注。氟苯尼考属动物专用广谱抗生素,具有抗菌活性高、毒副作用小等优点,主要用于牛、猪、鸡和鱼类等食品动物的细菌性疾病。因此,研究肠球菌对氟苯尼考的耐药机制,对于食品安全和人类健康具有重要的公共卫生学意义。本试验从不同来源(人、猪粪和猪病变内脏、生鲜肉和食品)分离、鉴定肠球菌149株。在此基础上,进行了肠球菌药敏试验,氟苯尼考耐药基因(cfr、fexA、fexB)的检测,质粒接合和电转化试验,耐药基因遗传环境分析,来探讨肠球菌对氟苯尼考的耐药机制。并通过毒力岛Marker基因esp的检测,细菌MLST分型等,来探讨不同来源粪肠球菌发生传播的可能性。结果显示,共分离鉴定到粪肠球菌72株,屎肠球菌40株。149株肠球菌对庆大霉素、四环素、环丙沙星、奎奴普丁/达福普汀、红霉素、氯霉素、氨苄西林、替考拉宁和氟苯尼考的耐药率分别为94%、91%、73%、70%、62%、44%、36%、10%和7%。对11株氟苯尼考耐药菌株进行耐药基因(cfr、fexA和fexB)检测,cfr均为阴性,fexA单阳性3株,fexB单阳性1株,fexA+fexB双阳性7株。质粒接合和电转化试验表明,携带fexA基因的质粒可以发生接合,携带fexB基因的质粒可以发生转化。fexA、fexB基因位于质粒上的特性有助于其发生水平转移。fexB基因的遗传环境分析显示,fexB基因位于一个新型的两端反向重复的插入序列内,该插入序列命名为ISEfa14。该插入序列形成的复合转座子功能有待于进一步试验证实。在人、猪和食品源粪肠球菌中检测到esp阳性菌株13株。其中,人源肠球菌7株均为ST4,1株为ST116;猪源肠球菌1株为ST4,1株暂未定型;食品源肠球菌2株ST16,1株ST69。猪源和人源粪肠球菌存在着一个共同的MLST型(ST4),表明猪源和人源粪肠球菌存在着传播的可能性,其耐药性和致病性值得进一步关注。综上所述,本研究调查了河南地区不同来源肠球菌(人、猪粪和猪病变内脏、生鲜肉和食品)的耐药状况。在此基础上,探讨了肠球菌对氟苯尼考的耐药机制,并新鉴定了一个插入序列ISEfa14,为fexB基因的水平扩散提供了部分理论解释。
[Abstract]:The characteristics of multidrug resistance of Enterococcus and the emergence of vancomycin resistant Enterococcus have attracted wide attention in medicine and veterinary clinic. Florfenicol is an animal specific broad-spectrum antibiotic with high antibacterial activity and low toxicity. It is mainly used for bacterial diseases in food animals such as cattle pigs chickens and fish. Therefore, the study of resistance mechanism of Enterococcus to florfenicol has important public health significance for food safety and human health. 149 strains of Enterococcus were isolated from different sources (human, pig manure, diseased viscera, fresh meat and food). On this basis, the drug sensitivity test of Enterococcus, detection of florfenicol resistance gene (cfr,fexA,fexB), plasmid conjugation and electrotransformation test, genetic environment analysis of drug resistance gene were carried out to explore the resistance mechanism of Enterococcus to florfenicol. The possibility of transmission of Enterococcus faecalis from different sources was studied by detecting esp of virulence island Marker gene and typing of bacteria MLST. The results showed that 72 strains of Enterococcus faecalis, 40 strains of Enterococcus faecium and 149 strains of Enterococcus faecium were isolated and identified as gentamicin, tetracycline, ciprofloxacin, quinaputin / dafopudine, erythromycin, chloramphenicol, ampicillin. The drug resistance rates of teicoplanin and florfenicol were 94 / 91 and 73 / 70, respectively. Eleven florfenicol resistant strains were detected by cfr,fexA and fexB. All of them were negative for fexA, 3 strains were single positive for fexB and 1 strain was double positive for fexA fexB. The results of plasmids conjugation and electrotransformation showed that plasmids carrying fexA gene could be conjugated. The character that the plasmid carrying fexB gene can be transformed. FexB gene is located on the plasmid. The genetic environment analysis of the horizontal transfer of the. FexB gene shows that the FexB gene is located in a new type of insert sequence with reverse repeat at both ends. The insert sequence is named ISEfa14. The function of the complex transposon formed by the insertion sequence needs to be confirmed by further experiments. Thirteen esp positive strains were detected in human, pig and food source Enterococcus faecalis. Among them, 7 strains of human Enterococcus were ST4,1 strains, 1 strain was ST116;, 1 strain was ST4,1 strain, and 2 strains of Enterococcus enterococcus from food were ST16,1 strain ST69.. There is a common MLST type (ST4) between porcine and human Enterococcus faecalis, which indicates that porcine and human Enterococcus faecalis can be transmitted, and its drug resistance and pathogenicity are worthy of further attention. To sum up, the drug resistance of Enterococcus from different sources (human, pig manure and diseased viscera, fresh meat and food) in Henan was investigated. On this basis, the drug resistance mechanism of Enterococcus to florfenicol was discussed, and a new insertion sequence ISEfa14, was identified, which provides some theoretical explanation for horizontal diffusion of fexB gene.
【学位授予单位】：河南农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.61

【相似文献】