RamA与MarA和SoxRS对沙门菌AcrAB-TolC外排泵表达的协同调控机制

Salmonella spp. are common zoonotic pathogen, and non-typhoidal Salmonella (NTS) are one of the most common causes of food and water borne infections, and the emergence and transmission of multi-drug resistant (MDR) strains that may pass from food animals to humans is a public health concern. The multi-drug resistant Salmonella from food animal in China is increasing, with high level of resistance to a broad spectrum of drug surpassing greatly that in developed countries. The multiple antibiotic resistance (mar) locus of E. coli and Salmonella is recognized as the most important intrinsic system, which mediate resistance primarily by up-regulating efflux of some antibiotics, disinfectants and organic solvents via the AcrAB-TolC efflux pump and down regulating influx through Outer Membrane Protein. Whilst the level of antibiotic resistance conferred by AcrAB-TolC is only moderate, but possibly as a 'stepping stone' to higher levels of resistance through MarA and RamA and SoxS. It is still unclear on the regulation between RamA and MarA, and RamA and SoxS on the expression of AcrAB-TolC. In this proposal, we focus on multi-drug resistant Salmonella spp. obtained from food animal, and step-wise selected with sub-inhibitory concentration or increasing concentration of ciprofloxacin,investigate the expression of RamA, MarA, and SoxRS by RT-PCR in strains with interest encoding gene knock-out, and eliminate the regulation of RamA, MarA and SoxRS on expression of AcrAB-TolC and cross talk between these globle regulators including RamA, MarA, SoxRS.

沙门菌是重要的人兽共患病病原，也是引起全球食源性疾病暴发的首要病原菌。目前沙门菌多重耐药现象日益严重。外排泵被认为是大肠杆菌和沙门菌多重耐药性的最重要的机制，其激活后能够将进入细胞内的药物进行非选择性的主动外排，导致细菌产生多重耐药性。AcrAB-TolC是革兰氏阴性菌中最主要也是研究得最为透彻的外排泵，其激活（表达）受局部和整体调控蛋白相互之间复杂、缜密的调控，其中RamA, MarA和SoxRS对AcrAB-TolC表达的调控作用尤其重要，它们可以独立或交互作用来调控AcrAB-TolC的表达。目前RamA与MarA和SoxRS之间如何协同调控AcrAB-TolC表达的机理还不明了。本项目拟针对养殖场分离和实验室诱导的耐药沙门菌，通过基因敲除的方法，构建目的基因缺失菌株，采用RT-PCR方法，探讨 RamA与MarA和SoxRS协同调控AcrAB-TolC表达的机制。

沙门氏菌是重要的食源性病原菌，对人类健康和公共卫生造成威胁。氟喹诺酮类是治疗沙门氏菌感染的一线药物，由于在医学临床广泛应用和动物养殖过程中促生长应用，导致耐药性的产生，给临床治疗带来困难。药物靶位突变和外排泵作用增强是沙门氏菌对氟喹诺酮类产生耐药的主要机制。本课题首先对我国养殖场沙门氏菌进行流行病学调查，揭示了流行的耐药（氟喹诺酮类、头孢菌素类）沙门氏菌的优势血清型为印第安纳型和鼠伤寒型，且印第安纳型耐药率高于鼠伤寒型。接着从临床分离的敏感的沙门氏菌中筛选出不同血清型菌株，建立外排泵及调控因子缺失菌，开展了环丙沙星选择压力下不同血清型沙门氏菌耐药发展机制的研究，尤其对acrAB-tolC外排泵及其他外排泵功能及调控进行了深入研究，并进一步探讨了优势血清型沙门氏菌适应性及毒力研究。研究结果表明，除了众所周知的ramA在调控沙门氏菌acrAB-tolC表达的作用外，首次揭示了marA和ramA对acrAB-tolC自身反馈调节、acrAB-tolC与其他外排泵相互表达起到共调节作用；首次揭示了印第安纳型沙门氏菌在环丙沙星选择压力下具有较高突变频率，易于发展成耐药菌株；环丙沙星选择压力下，获得耐药表型的鼠伤寒沙门氏菌生长能力明显强于肠炎型。野生型鼠伤寒沙门氏菌比野生型肠炎沙门氏菌具有生长竞争优势，但耐药性发生初期，肠炎沙门氏菌较具有竞争优势，但是一旦获得对环丙沙星的耐药性，鼠伤寒沙门氏菌翻转成优势菌株。说明环丙沙星耐药的鼠伤寒沙门菌比对肠炎沙门菌更具有适应性优势。在低胆盐环境中肠炎型沙门氏菌侵袭力强于鼠伤寒型，但在高胆盐情况下，耐药发展初期敏感性降低的鼠伤寒沙门菌的侵袭能力远远强于肠炎沙门菌，获得耐药表型后两者的侵袭能力都大大降低。. 本课题首次创新性地从血清型角度探讨沙门氏菌耐药发展机制及适应性，研究结果对现有的沙门氏菌耐氟喹诺酮类耐药机制做了非常重要的补充，也阐释了我国养殖场流行沙门氏菌的特征及其原因，研究结果对临床合理用药、控制沙门氏菌的传播扩散都具有重要的指导意义和理论意义。