The Regulatory Mechanism Of BasS/BasR Two-component System In Avian Pathogenic Escherichia Coli Drug Resistance

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,2.5?g/mL,12.5?g/mL,2.0mg/mL,MG1655 and MG 1655?basSR minimal inhibitory concentrations of Erythromycin,Norfloxacin and Doxycycline were l.2mg/mL,400mg/mL,2.5?g/mL,3.125?g/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.