P1/P1-like噬菌体介导的mcr-1基因水平转移及其分子机制

The mcr-1 gene could mediate resistance to the last resort antibiotic in human medicine—colistin. This gene posed a great threat to the clinical therapy and public health after its occurrence and prevalence in human and animals. Up to now, most investigators mainly focused on the role of plasmid and insertion sequences to the transmission of mcr-1 and no one paid attention to the phage. However, we recently found that most of the mcr-1-positive Escherichia coli from pigs carried the P1/P1-like phage elements. Considering that the phage is the bacterial-dependent virus and could act as the mobile element between bacteria, a hypothesis is proposed that the phage may play a role to the transmission of mcr-1 gene. To prove this hypothesis, we isolated the mcr-1-positive E. coli harboring the P1/P1-like prophage from pig farms, and then the prophage was induced and characterized. The mcr-1 gene was transducted by P1/P1-like phage and fitness cost was studied. Whole genome sequencing and comparative genomics were used to analyze the critical genetic elements that were involved in the horizontal transfer of mcr-1 gene. Findings in this study will reveal a novel transmission mechanism of mcr-1 gene as well as provide scientific basis for the risk assessment and resistance containment in pig farms.

多粘菌素耐药基因mcr-1在人源和动物源细菌中的出现与流行，已成为全球严峻的临床及公共卫生问题。目前对mcr-1基因的分子传播机制研究主要集中于质粒或插入序列上，而很少关注噬菌体在该基因水平转移中的作用。申请人近期发现，国内某些猪场分离的mcr-1阳性大肠杆菌大多携带P1/P1-like噬菌体序列，考虑到噬菌体作为细菌寄生病毒和穿梭于细菌间的另一个“移动元件”，推测P1/P1-like噬菌体可能在该基因的传播过程中发挥作用。本研究拟通过分离猪场中携带P1/P1-like前噬菌体的mcr-1阳性大肠杆菌，结合溶原性噬菌体的诱导、体外转导试验、适应性代价研究、全基因组测序技术和比较基因组学方法来阐明猪源大肠杆菌P1/P1-like噬菌体介导mcr-1基因水平转移的分子机制。该研究结果将揭示mcr-1基因传播扩散的新途径和新机制，为猪源mcr-1基因转移扩散的风险评估和防控提供重要科学依据。

mcr-1 基因可介导人医临床抗生素最后一道防线—多粘菌素的耐药，该基因在人源和动物源革兰氏阴性细菌中的出现和流行，已成为全球严峻的临床及公共卫生问题。目前绝大部分报道表明 mcr-1 基因定位于细菌质粒上，通过接合转移或插入序列介导 mcr-1 基因在菌株和菌种间水平传播，而研究者很少关注噬菌体在 mcr-1 基因水平转移中的作用。为了明确P1-like 噬菌体在mcr-1 基因水平转移中的作用，本项目分离筛选了多株携带P1-like 噬菌体型质粒的猪源大肠杆菌。基因定位实验表明mcr-1 基因全部定位于P1-like 噬菌体型质粒上，该质粒不携带其它任何耐药基因，且不具有接合转移特性。通过紫外线或丝裂霉素进行诱导，发现P1-like噬菌体型质粒不能够从宿主菌中诱导出来，因此不能够通过转导机制介导mcr-1 基因水平转移。基因环境分析表明P1-like噬菌体型质粒上有关噬菌体活性的一些重要结构蛋白基因的缺失可能是导致P1-like噬菌体不具有诱导活性的重要原因。部分P1-like噬菌体型质粒上，mcr-1基因的两侧存在同向的ISApl1插入序列，可通过同源重组形成环状中间体结构从质粒上游离下来，因此具有水平转移mcr-1基因的风险。除此之外，研究中从猪源大肠杆菌中发现了一种新型的替加环素耐药基因tet(X4)，该新型耐药基因可与mcr-1基因共存于同一株大肠杆菌中，并且接合型的tet(X4)质粒可以辅助mcr-1阳性的P1-like 噬菌体型质粒进行水平转移。以上研究结果明确了猪养殖场中P1-like 噬菌体型质粒对于mcr-1基因传播的作用，揭示了mcr-1 基因传播的一种新机制，从而为江苏省猪源大肠杆菌中 mcr-1 基因传播的风险评估和防控提供科学依据。