【作者】 李静；

【导师】 薛挺；

【作者基本信息】 安徽农业大学 ， 微生物学， 2019， 硕士

【摘要】 禽致病大肠杆菌(Avian pathogenic Escherichia coli,APEC)是严重危害我国及世界上禽畜养殖业中常见的致病菌之一,尤其在鸡、鸭养殖业最为常见,因其发生及分布范围广,目前为止对于防治禽致病大肠杆菌病,除了大量使用抗菌药物外依然没有更好的办法。随着抗生素的长期使用以及滥用,导致耐药谱不断扩大,不仅造成其耐药性不断增强,而且还出现了多重耐药菌株。因此,寻找新的药物靶点十分重要。双组分调控系统是广泛分布于各种细菌体内的一种调控系统,BasS/BasR是大肠杆菌内一种信号转导系统。但在多重耐药菌株中,BasS/BasR是否与其耐药性有关联,学术界鲜有报道。转录组学的结果表明多重药物外排泵emrD的转录水平在basSR敲除株中发生了变化。此实验揭示了 BasS/BasR双组分系统对禽致病大肠杆菌耐药性的影响,进而说明BasSR双组分系统对耐药性的调控机制。通过转录组学分析,发现basSR基因敲除株的多个药物外排泵编码基因的转录水平与野生型菌株相比有显著性差异。1.为了验证野生株和敲除株的耐药程度,选用二倍稀释法对临床分离株APEC40、APEC40ΔbasSR 和 MG1655、MG1655ΔbasSR 进行耐药性实验。结果发现,APEC40和APEC40ΔbasSR对克林霉素、环丙沙星、四环素、多西环素和十二烷基硫酸钠的最小抑菌浓度分别为 1.0mg/mL、2.5μg/mL、12.5μg/mL、2.0mg/mL,MG1655 和MG1655ΔbasSR对林可霉素、红霉素、诺氟沙星和多西环素的最小抑菌浓度分别为1.2mg/mL、400μg/mL、2.5μg/mL、3.125μg/mL,说明basSR基因敲除后,APEC40ΔbasSR和MG1655AbasSR耐药性均有所降低。2.为进一步研究basSR基因敲除对菌株的影响,选用细菌存活率和比浊法对其进行研究,结果表明,当basSR基因敲除后,basSR基因敲除株表现出了对喹诺酮类、四环素类、林可霉素类及大环内酯类等抗菌剂的耐药性变化,说明BasSR系统对禽致病大肠杆菌的耐药性存在调控效应。3.通过RNA抽提、反转录PCR实验和实时荧光定量PCR(RT-qPCR)实验对耐药基因emrD的转录水平变化进行检测。结果发现,当basSR基因敲除后,敲除株的emrD基因转录水平与野生株相比,其转录水平不同程度的有所降低,而在回补菌株中,得到了显著的回补。说明basSR基因敲除后对emrD基因转录水平有显著的影响。4.关于BasS/BasR系统对耐药基因的调控研究,利用蛋白体外表达与纯化技术、蛋白-DNA凝胶阻滞技术等分子生物学手段研究BasR蛋白对emrD耐药基因的调控,确定直接调控或间接调控的调控类型。实验结果表明,BasR蛋白可以与emrD耐药基因的启动子区域发生特异性的结合。由此推断,BasSR系统是依赖于BasR蛋白直接调控耐药基因emrD的转录。该研究也为寻找防治禽致病大肠杆菌的潜在药物靶标以及探寻辅助抗生素治疗APEC感染的新思路提供重要的实验基础和科研依据。更多还原

【Abstract】 Avian pathogenic Escherichia coli is one of the most common pathogenic bacteria in livestock industry in China and the world,especially in chicken and duck breeding,because of its wide range of occurrence and distribution.The high morbidity and mortality not only seriously restrict the development of poultry breeding industry,but also there is no better way to prevent and cure avian pathogenic colibacillosis in addition to the use of antibiotics.With the long-term use and abuse of antibiotics,the resistance spectrum is expanding and the rate of variation is increasing,which not only results in the increase of drug resistance,but also the emergence of muti-drug resistant strains.Therefore,it is very important to find new drug targets.Two-component system is a kind of regulation system widely distributed in various bacteria,and BasS/BasR is a signal transduction system in Escherichia coli.However,it is rare to report whether BasS/BasR is associated with drug resistance in multi-drug resistant strains.The results of transcription histology showed that the transcription level of multi-drug efflux pump emrD was changed in basSR knockout strain.This experiment revealed the effect of BasS/BasR two component system on drug resistance of avian pathogenic Escherichia coli,and further demonstrated the mechanism of BasSR two-component system regulating drug resistance.The transcription level of several drug efflux pump coding genes of basSR knockout strain was significantly different from that of wild strain by means of transcriptome analysis.1.In order to verify the drug resistance of wild and knockout strains,the method of two times dilution was used to assess Escherichia coli strains of clinical isolates APEC40,APEC40AbasSR and MG 1655,MG 1655△basSR.The results showed that the minimal inhibitory concentrations of APEC40 and APEC40AbasSR,Clindamycin,Ciprofloxacin,Tetracycline,Doxycycline and SDS were 1.0 mg/mL,2.5μg/mL,12.5μg/mL,2.0mg/mL,MG1655 and MG 1655△basSR minimal inhibitory concentrations of Erythromycin,Norfloxacin and Doxycycline were l.2mg/mL,400mg/mL,2.5μg/mL,3.125μg/mL,respectively.2.In order to further study the effect of basSR gene knockout on wild type,the survival rate and growth curve method were used to study it.The results showed that the basSR gene knockout strain showed quinolones after basSR gene knockout.The changes of drug resistance of tetracycline and macrolides suggest that BasSR has a regulatory effect on the resistance of avian pathogenic Escherichia coli.3.The transcription level of emrD gene was detected by RNA extraction,reverse transcription PCR assay and real-time quantitative polymerase chain reaction(RT-qPCR)assay.According to the level of transcription of the related genes,the functional genes connecting the BasSR system with drug resistance were finally determined.4.To study the regulation of drug resistance gene by BasS/BasR system,the regulation of BasR protein on emrD resistance gene was studied by molecular biological methods such as protein expression and purification in vitro,protein-DNA gel block technique and so on.Determine the type of regulation of direct or indirect regulation.This study provides an important experimental basis and scientific basis for finding potential drug targets for the prevention and treatment of avian pathogenic Escherichia coli and exploring new ideas of adjuvant antibiotics in the treatment of APEC infection.更多还原