猪源产NDM-1细菌的分离鉴定及其人-猪交互传播可能性研究

The emergence of drug-resistant super bacteria which produce NDM-1, pose a great threat to human health. These bacteria have become resistant to almost all known and useful antibiotics. Since found in human Acinetobacter baumannii for the first time in 2009, such bacteria were found in groups from other human pathogens, normal carrying flora, and even environmental microorganisms. But so far, there is no report about detecting such NDM-1-producing strains in animals at home and abroad. Using a self-designed experimental program, 3 swine Escherichia coli strains containing blaNDM-1 gene were first isolated from pig faeces. On this basis, the subject will detect the distribution of NDM-1 bacteria in the swine, compare the homology of blaNDM-1 plasmids from pigs and the human at the molecular level, find the swine/human transfer characteristics of blaNDM-1 plasmids and invasion characteristics of the pig blaNDM-1 host strain to human cells. Finally, we will comprehensively assess the potential risks of pig source blaNDM-1 gene in pigs and people. This study will provide an important reference for the control of the NDM-1-producing bacteria, public health and the safety of animal original food.

产NDM-1细菌是一类新型细菌，几乎对所有抗生素都有耐药性，给人类健康带来极大威胁。自2009年首次被发现以来，从人类病原菌群、正常菌群甚至环境微生物中均发现了该类细菌。但是，目前均没有在动物体内检出产NDM-1细菌的报道。申请人从猪的粪便样本中成功分离到3株产NDM-1大肠埃希氏菌并初步研究了其耐药基因特征。本项目将进一步扩大样本量，研究产NDM-1细菌在中国猪群中的分布情况及规律。然后，对其耐药质粒进行全基因组测序和注释，解析猪源NDM-1基因及其载体质粒的分子特征；同时与人源NDM-1+质粒进行比较分析，阐释其同源性、亲缘关系和进化地位。最后，对猪源NDM-1+质粒在猪源/人源细菌间的转移特性，猪源产NDM-1细菌对人源细胞的侵袭能力等进行鉴定，评估其在猪群内部的扩散能力及发生人-猪互传的潜在风险。本项目将为猪源产NDM-1细菌的防制、公共卫生和保障动物源性食品安全提供重要参考依据。

产NDM-1细菌是一类新型细菌，几乎对所有抗生素都有耐药性，给人类健康带来了极大威胁。本课题从不同猪群的感染病例中分离出13株产NDM-1大肠埃希菌和1株鲍曼不动杆菌，并对其中2株产NDM-1大肠埃希菌（E120413株和E131225株）的生物学特性进行了系统研究。药敏试验结果显示，这2株细菌对大观霉素、四环素和呋喃唑酮中度敏感，对阿莫西林等其他15种抗生素均耐药。最小抑菌浓度试验表明，2株菌对厄他培南、美罗培南等碳青霉烯类抗生素具有很强的耐药性。2株细菌均能扩增到281 bp的特异性blaNDM-1基因片段。产NDM-1大肠埃希菌的仔猪感染特性研究结果表明，E120413株细菌能从接种仔猪快速传染给未接种的同居组仔猪。仔猪血液样本的检测结果表明，E120413株细菌能快速突破仔猪的免疫防御进入血液循环形成菌血症。但仔猪的临床症状与病理学检查结果表明，E120413并不能导致猪的明显临床症状和严重病理变化。这些试验结果显示，产NDM-1大肠埃希菌E120413株对猪具有较强的传播扩散能力和持续感染能力，但其致病力较弱。然后，我们又通过动物感染试验研究了猪源产NDM-1大肠埃希菌E120413株对豚鼠、小鼠和雏鸡的跨种感染特性。这些试验结果表明，E120413株对豚鼠、小鼠和雏鸡的感染能力均弱于对猪的感染能力，其持续感染能力强弱依次为：猪> 小鼠> 豚鼠> 鸡。猪源产NDM-1大肠埃希菌耐药质粒的转移特性研究结果表明，E120413株和E131225株细菌均能把自身携带的blaNDM-1质粒接合转移到9种受体菌中，具有很强的耐药质粒扩散能力。采用大质粒的PE库高通量测序策略，对猪源E120413株的blaNDM-1+质粒进行全基因组测定和序列解析，对其blaNDM-1的分子特征进行了阐释。该研究评估猪源产NDM-1细菌在猪群内部的扩散能力及发生人-猪互传的潜在风险和保障动物源性食品安全具有重要意义。