水产源磺胺耐药基因sul1、sul2和sul3 在大肠杆菌中的适应性代价及代偿机制研究

Sulfonamides are widely used in aquaculture industry in China, and the resulting sulfonamide-resistant bacteria and drug-resistant genes sul1, sul2 and sul3 have a high prevalence in aquaculture and the environments. However, the fitness costs and compensatory mechanisms of the three drug-resistant genes in related host bacteria are unclear, which severely restricts the understanding of their dissemination mechanisms. Therefore, our project will first reveal the in vitro fitness costs of the three drug-resistant genes in E. coli among different external environments, such as aquaculture, through the construction of drug-resistant engineering bacteria, in vitro competition experiments and flow cytometry. Then, in vivo competition experiments were conducted on the intestinal tracts of female BALB/c mice by using drug-resistant engineered bacteria in order to determine in vivo fitness costs of the three drug-resistant genes in E. coli. Finally, through the colonization test of the drug-resistant engineering bacteria in the mice intestinal tracts, the fitness compensatory recovery strains of the three drug-resistant genes in the intestinal tracts were obtained, and then the possible compensatory mechanisms were elucidated through quantitative proteomics and qPCR technology. It is helpful to further reveal the reasons for the prevalence of sul1, sul2 and sul3 genes in aquatic products and related environments, and to evaluate the potential of their continued proliferation and colonization into animal (human) intestinal tracts, which providing theoretical basis for the risk assessment and prevention and control technology of antibiotic resistance of food-borne pathogenic bacteria.

磺胺类药物在我国水产养殖业中应用广泛，由此产生的磺胺耐药菌及耐药基因sul1、sul2和sul3在水产及其环境中流行率高。然而，3个耐药基因在相关宿主菌中的适应性代价和代偿机制不明，严重制约了对其传播机制的深入理解。因此，本项目将通过耐药工程菌构建、体外竞争试验法和流式细胞术等方法揭示携带磺胺耐药基因的大肠杆菌在水产养殖等外界环境中的体外适应性代价。然后利用耐药工程菌在雌性BALB/c小鼠动物肠道进行体内竞争试验，明确其在小鼠肠道的体内适应性代价。最后通过耐药工程菌在小鼠肠道的定殖试验，获得其在肠道内的适应性代偿回复株，进而通过定量蛋白组学和qPCR技术，阐明其在动物肠道内发生适应性代偿的可能机制。以上内容的完成有助于进一步揭示sul1、sul2和sul3基因在水产及相关环境中流行的原因，并评估其继续扩散和定殖到动物（人体）肠道中的潜力，为食源性致病菌耐药性的风险评估和防控提供理论依据。

本研究围绕食源性病原微生物耐药基因传播这一全球共同关注的食品安全和人类健康热点问题，以本项目组前期分离鉴定到的水产源大肠杆菌磺胺耐药基因sul1、sul2和sul3为研究对象，旨在阐明其在大肠杆菌中的适应性代价和代偿机制，以期为进一步揭示磺胺耐药基因水平传播机制提供理论依据。本研究构建了遗传背景一致的分别携带sul1、sul2和sul3基因的大肠杆菌工程菌株，分别进行体外和体内适应性代价试验，结果显示：携带sul1、sul2或sul3基因的大肠杆菌工程菌株在体外环境适应性均较好，但3株菌在贫营养培养条件下均存在显著的适应性代价 (p < 0.05)，而添加亚抑制浓度的磺胺异恶唑后菌株适应性得到显著回复 (p < 0.05)。此外，3株菌在体内环境均有一定的适应性代价，但携带sul2基因的大肠杆菌适应性代价得到回复，且在第9天筛到3株代偿株；进而利用Tandem Mass Tag定量蛋白组学技术和 Parallel Reaction Monitoring靶向定量技术在蛋白水平上探索磺胺耐药基因表达时大肠杆菌适应性代偿机制，结果显示：（1）双组分系统中，代偿株常通过调节关键蛋白的表达量，来提高自身运动能力、黏附力和侵入肠细胞的能力，从而提升适应性；（2）代偿株可能通过优化半乳糖代谢通路相关蛋白的表达，对自身糖原合成和细胞壁合成进行更精细的调控，从而从细胞能量供给和维持细胞形态等方面提高自身在肠道中的适应性；（3）代偿株的TnaA和TnaB蛋白表达量均显著上调 (p < 0.05)，TnaA上调倍数是所有蛋白中最高的，使得胞内吲哚浓度升高，菌株进而通过积累较高浓度吲哚来调节自身的趋化性和适应性，增加菌株在肠道定植水平和适应能力。本研究结果加深了对磺胺耐药基因传播的认识，同时为探寻其污染防控技术提供了科学理论依据。