The Key Active Efflux Pump Genes Of Baicalin Increase Susceptibility Of Ciprofloxacin

In animal husbandry industry,Escherichia coli is one of the most common and high incidence pathogens,It can cause urinary system,respiratory system infections of domestic animals and human.With the recent abuse of antibiotics,E.coli resistance to commonly used antibiotics has reached a very serious trend,active efflux system is one of its main resistance mechanism.Efflux pump inhibitors can restore the antibacterial activity of common antibiotics and reduce the drug resistance rate.Previous studies have shown that baicalin is a potential efflux pump inhibitor could enhances ciprofloxacin.The purpose of this study is to study baicalin could sensitize the main efflux gene of ciprofloxacin,to excavation of the potential of the old drug and overcome the single drug.It is better applied to veterinary clinical lay the foundation for ciprofloxacin.1 In order to explore the effect of baicalin combined with ciprofloxacin,this experiment carried out micro-broth dilution method,in this study the resistance of 295 animal-source E.coli to quinolones were determined,the results showed that 61.7%-72.2% of E.coli isolates presented resistance to 4 different quinolones,and the highest of the total drug-resistance rate was ciprofloxacin.The total number of the strain was 8 which MIC of Ciprofloxacin ?64?g/mL.The MIC of baicalin to the above 8 strains,2 for 512?g/mL,6 for 1024?g/mL;The protocol was designed by checker board method,to investigate the combination of baicalin and ciprofloxacin,and the result showed that the two drugs have synergistic effect,FIC index were 0.375.The 1/16,1/8 MIC baicalin can significantly reduce the MIC of ciprofloxacin,thus determining the optimum concentration of baicalin in combination with ciprofloxacin at a concentration of 32?g/mL.2 To investigate the effect of baicalin on mRNA expression of active efflux gene of Escherichia coli,the above eight resistant strains were treated in the following five ways:(1)without any drugs(2)add 32?g / mL baicalin(3)4?g / mL ciprofloxacin(4)32?g / mL baicalin and 4?g / mL ciprofloxacin 16(5)?g / mL CCCP and 4?g / mL ciprofloxacin,extracting total RNA and reverse transcription cDNA,RT-PCR.measuring the Ct values of active efflux regulatory genes and housekeeping gene gap A,and the mRNA expression of acrA,acrD,marA,acrE,acrF,acrB,mdt A,robA,sox S were calculated under different culture conditions.The results showed that the expression of acrE,acrB,acrF,mdt A,robA,sox S gene was not obvious when baicalin and ciprofloxacin combined with ciprofloxacin alone,but acrA,acrD,marA gene expression were significantly decreased,suggesting that these three genes are the target of baicalin.3 To investigate antisense oligonucleotides silencing acrA,marA,acrD genes,before and after comparison effects of baicalin on susceptibility of ciprofloxacin to Escherichia coli.We desingned primers of three genes,seted the blank control group,baicalin group,antisense oligonucleotide silencing group,antisense oligonucleotide silencing + baicalin group.They were transfected into the competent cells of clinical drug-resistant bacteria and were coated on a certain concentration of ciprofloxacin.Then the growth of colonies was observed and counted.The results showed that acrA,marA were silenced,baicalin to the sensitivity of ciprofloxacin was not obvious;after the acrD gene was silenced,the sensitivity of baicalin to ciprofloxacin was decreased.The above results indicate that acrA,marA play a major role in the antibacterial sensitization of baicalin,and acrD plays a minor role.