Research On The Antibacterial Effect Of Chelerythrine Against Methicillin-resistant Staphylococcus Aureus

Staphylococcus aureus is an important gram-positive pathogen that can infect humans,livestock,poultry,and various wild animals.Due to the abuse of antibiotics,drug-resistant Staphylococcus aureus has gradually increased,among which MRSA has caused significant harm to public health safety at home and abroad.Chelerythrine（CHE）is a natural chinese herbal compound with antibacterial,heat clearing,detoxification,and other effects.The study shows that CHE has antibacterial activity against MRSA,but its antibacterial mechanism is not yet clear.Therefore,the aim of this study is to elucidate the antibacterial effect of chelerythrine against MRSA,focusing on analysis of the antibacterial mechanism,and providing new theoretical reference for clinical prevention and treatment of methicillin-resistant Staphylococcus aureus infection.Microdilution method and kinetic bactericidal curve were used to determine the antibacterial activity of CHE against MRSA.The hemolytic activity of CHE on rabbit red blood cells was detected by ultraviolet spectrophotometry.Using NPN,extracellularβ-galactosidase activity test,and DISC₃（5）to detect the cell wall permeability,membrane permeability,and proton-motive force of CHE against MRSA strain.The ultrastructural changes of Staphylococcus aureus induced by CHE were studied using scanning electron microscopy and transmission electron microscopy.The results showed that the minimal inhibit concentration（MIC）of CHE against Staphylococcus aureus was 6.25μg/m L,minimal bactericidal concentration（MBC）is 12.5μg/m L,and has a dose dependent inhibitory effect on the growth of the strain.The results showed that CHE could increase the permeability of the cell wall and membrane of the strain,destroy the proton-motive force inside and outside the cell membrane,cause cell shrinkage and collapse,reduce electron density,and cause content leakage.Moreover,high concentrations of CHE still do not exhibit hemolytic activity.The gene expression changes of the mechanism of CHE on methicillin resistant Staphylococcus aureus were analyzed using transcriptomics technology.Transcriptome sequencing results showed that there were 1102 differential genes causing significant changes in methicillin resistant Staphylococcus aureus ATCC43300 caused by CHE,including 609 significantly upregulated genes and 493 significantly downregulated genes.Differential genes showed that CHE treatment resulted in decreased expression of DNA helicase Dna B and primer enzyme Dna G,transcription related rpo BC genes,ribosomal synthesis rps DH genes,respiratory chain sdh ABC genes,ATP synthase atp AH genes,β-lactam resistance related genes,and vancomycin resistance related genes in the strain.Fluorescence quantification was used to validate partial predicted targets.The results showed that rpl AC,sdh AC,cyd A genes were significantly down regulated,and kdp B was significantly up regulated,which was consistent with the results of transcriptome.DCFH-DA fluorescence probe method was used to detect the effect of CHE on the ROS production of the strain.The results showed that CHE could rapidly increase the ROS level in ATCC 43300 cells.Through molecular docking analysis,CHE may exert its activity by binding to proteins such as SDH.Through chemical colorimetric analysis,it was found that CHE can reduce the SDH,T-AOC,CAT activity of the strain,and increase MDA content.In conclusion,the above shows that CHE may be to inhibit the formation of ribosome and block the electronic transmission of respiratory chain,and promote ROS production by inhibiting the expression of ribosome protein gene rpl AC and sdh ABC and other genes,resulting in oxidative stress of strains,exerting activity against MRSA.