Research On The Antibacterial And Sensitizing Activity Of Aqueous Extract Of Radix Paeoniae Rubra And Paeoniflorin On Drug-resistent E. Coli

Avian multiple antibioticresistant Escherichia coli caused a big loss to the aviculture.Radix Paeoniae Rubra of Qingre traditional Chinese medicines have broad-spectrum antimicrobial activity,and it can combine antibiotics(additive effect or synergistic action)to inhibit the drug-resistant E.coli,which has been confirmed by research.This study explored the antibacterial sensitization mechanism of paeoniflorin,the main component of Radix Paeoniae Rubra,from the perspective of multidrug resistant efflux pumps.To explore the mechanism of drug resistence elimination of Radix Paeoniae Rubra on avian multiple antibioticresistant E.coli by combining transcriptome and IBT quantification proteomics analysis.The bacteriostatic effect of Aqueous Extract of Radix Paeoniae Rubra and paeoniflorin of its main component combined with antibiotic on clinically isolated the multiple antibiotic resistant avian Escherichia coli E320 and E.coli ATCC35218 of quality-controlling strain was studied by checkerboard microdilution method.The results confirmed that the combination of Aqueous Extract of Radix Paeoniae Rubra,paeoniflorin and fosfomycin Sodium has synergistic effect on the inhibition of the multiple antibiotic resistant Escherichia coli E320(FICI?0.5),and Aqueous Extract of Radix Paeoniae Rubra combined with ceftriaxone sodium showed the additive effect on E320(0.5<FICI?1),and the combination of Aqueous Extract of Radix Paeoniae Rubra and fosfomycin Sodium has synergistic effect on the inhibition of E.coli ATCC35218(sensitive strain of fosfomycin sodium).Using YASARA software,targeting the efflux pump transporter AcrB in the multiple antibiotic resistant AcrAB-TolC system of E.coli.Molecular docking of AcrB and paeoniflorin.Molecular docking results showed that the binding energy of paeoniflorin and AcrB protein receptor reached 8.333 kJ/mol.The main sites of action were Phe628,Phe615,Val612,Ile277,Asn274,Gly179,Phe178 and other amino acid residues on the AcrB protein receptor.The main inner-molecular force is hydrophobic interaction.The efflux test of Nile red confirmed that paeoniflorin can inhibite the activity of efflux pump.Through Quantitative Real-time PCR,it was found that paeoniflorin combined with fosfomycin sodium can significantly down-regulate the expression of AcrB and the positive regulation genes SoxS,Rob,Fis,SdiA,RamA,RpoS mRNA,thereby reducing drug resistance and sensitizing bacteria.1/2 MIC(125 mg/mL)Aqueous Extract of Radix Paeoniae Rubra reduced the MIC of multi-drug resistant E.coli E320 to fosfomycin sodium from 512 ?g/mL to 256 ?g/mL.Through the combined analysis of transcriptome and proteome,it was found that there were 7 genes significantly changed in RNA-seq,but there was no significant change in proteomic analysis.In the proteome,the class of response to stimulus significantly down-regulated protein is the lethality reduction protein(yibA),class I fumarate hydratase.The class of cell part and membrane part were significantly down-regulated protein,including hydrogenase 2 small subunit,inner membrane protein of BrbK family alternate lipid exporter(YhjD),zinc-type alcohol dehydrogenase-like protein,inner membrane protein of DUF554 family(YqgA),arabinose transporter of MFS family(AraE).These proteins involve multiple functional categories and pathways.It is speculated that the reason for the elimination of drug resistence of E coli by the Aqueous Extract of Radix Paeoniae Rubra is mainly to significantly down-regulate the lethality-reducing protein(yibA),which makes the bacteria sensitive to stressors such as antibiotics,and significant down-regulation of the small subunit of hydrogenase 2 affects the energy source of the proton driving force of the drug-resistant efflux pump of bacteria.The antibacterial sensitization mechanism of paeoniflorin of the main component of Radix Paeoniae Alba on avian E.coli was verified by efflux pump test.Through the combined analysis of transcriptome and proteome,the mechanism of drug resistance elimination of Aqueous Extract of Radix Paeoniae Rubra to avian E.coli was revealed.To provide a theoretical basis for the further application of this class of drugs.