禽源耐碳青霉烯类大肠杆菌耐药性流行特点及其耐药机制研究

Carbapenem-resistant Escherichia coli（CRE） can resistance to almost all β-lactam antibiotics and have the ability to integrate other resistance genes.The emergence of this multi-resistant bacteria has caused great concern in human and veterinary clinical. The objetctives of this study were to evaluate the prevalance of CRE colonization in avians and avian farm staffs in China and to analyze the relationship of CRE from human and avians through Pulsed- Field Gel Electrophoresis (PFGE).At the same time, the CRE isolates were performed on other genotypic analysis Furthermore, the new mechanism of avians s source of carbapenem -resistant stapylococcus aureus were studied through electrotransformation, plasmid curing, plasmid or whole genome complete sequcencing, bioinformatic analysis, new potential gene cloning and functional identification. In conclusion, this research will be comprehensive reveal the characterization of CRE from avians and its relation of CRE isolates from avian farm staffs. The acqusition of mechanism of avians source of carbapenem-resistant staphylococcus aureus will provide the theoretical basis to further control the spread of CRE.

耐碳青霉烯类抗生素大肠杆菌(CRE)能够对几乎所有β-内酰胺类抗菌药物耐药，而且还具有整合其它耐药基因的能力。这种多重耐药菌的出现与流行已经引起了人医和兽医临床的高度关注。本项目通过调查我国东北地区禽源和工作人员CRE的流行特点，应用PFGE技术分析两种来源CRE间的亲缘关系，同时采用基因分型技术和国外流行株进行比较研究。另外，对出现的禽源大肠杆菌对碳青霉烯类抗生素耐药机制展开研究，采用电转化、质粒消除试验、质粒或染色体的基因组全测序、生物信息学软件分析、潜在新基因克隆和功能鉴定等方法获得新耐药基因。上述研究能更系统、全面的揭示我国东北地区禽源CRE的流行特点及与工作人员CRE的关系，同时可获得大肠杆菌介导的耐药基因，这为进一步防止CRE的流行和扩散提供了理论基础。

本研究从东北地区多个养殖场分离鸡源大肠杆菌，共鉴定成功413株。采用K-B法测定各分离菌株对22种抗生素的敏感性。结果表明鸡源大肠杆菌对头孢噻吩、氨苄西林和四环素耐药率较高，达80%以上；对阿米卡星、氨曲南和多黏菌素B高度敏感，敏感率达60%以上。对413株鸡源大肠杆菌进行碳青霉烯类耐药基因的检测。结果表明，NDM的检出率为0.24%，SPM的检出率0.48%、VIM的检出率0.48%、IMP和KPC均未检出。I类整合子检出率71.91%，整合酶I的检出率为65.52%，整合酶II的检出率为54.48%，未检出整合酶III。通过序列分析被检菌株中共有6种Ⅰ型整合子，其携带有6种不同基因盒的组合方式：aadA1-aadA22-aadA23、aadA1-aadA2-dfrA1-dfrA12-drfA15、aadA5-dfrA17、aadA1-aadA2-aadA29-dfrA1-dfrA12-dfrA15、aadA2-aadA28-dfrA12和aadA2-dfrA16。PCR扩增结果表明gyrA、gyrB、parC、parE阳性率分别为25.91%、53.03%、48.67%、63.68%。通过对PCR产物的进一步研究分析表明，gyrA亚基发生Leu55→Pro，Ser83→Leu，Asp87→Gly，Phe109→Leu和Ala136→Val突变。gyrB发生Pro52→Ser，Cys58→Tyr和Leu99→Pro突变。parC亚基发生Ser58→Ile，Gln62→Lys，Val122→Ala，Thr147→Ala和Val284→Met突变。parE 发生Gln40→Arg突变。鸡源大肠杆菌的ESBLs基因。350株大肠杆菌为产ESBLs株，占84.75%。PCR扩增表明TEM、CTX-M、SHV、OXA阳性率分别为58.11%、47.22%、29.78%、65.86%。为了解磺胺类、氯霉素类、四环素类、大环内酯类和氨基糖苷类药物的耐药基因的流行情况及其耐药机制，以便更加合理的用药。PCR扩增结果显示：sulⅠ、sulⅡ、sulⅢ的阳性率依次为46.00%、69.49%、44.31%；cmlA、cat、floR的阳性率依次为31.72%、29.54%、40.44%；tetB、tetC、tetD、tetM的阳性率分别为4.60%、11.62%、0.73%、2.18