Antibacterial Activity And Mechanism Of Syringic Acid Against Multidrug-resistant Staphylococcus Aureus

With the widespread use of antibiotics,bacterial resistance continues to increase,and "super bacteria" have become the focus of global attention.The incidence of multidrug-resistant Staphylococcus aureus isolates is increasing globally,which poses a severe global challenge to the treatment of Staphylococcus aureus-induced infections.Therefore,it is urgent to find a drug that can effectively inhibit multi-drug-resistant Staphylococcus aureus.In the development of new antibacterial drugs,traditional Chinese medicine is regarded as a valuable asset in the research of new antibacterial agents.The active ingredient syringic acid in the Chinese medicine Malan is a natural phenolic compound,which showed good antibacterial ability in the field of biomedicine.In this study,Sau-06 multidrug-resistant Staphylococcus aureus was selected as a test strain to explore the antibacterial activity of syringic acid on it and its bacteriostatic mechanism.175 strains of clinically-derived Staphylococcus aureus were used as the sample library.Multi-drug resistant Staphylococcus aureus with multi-drug resistance,carrying enterotoxin gene and strong biofilm formation ability was screened from 175 strains of Staphylococcus aureus by drug sensitivity test,Congo red test,crystal violet test,PCR test.And it was named Sau-06 multi-drug resistant Staphylococcus aureus as the subject.8 kinds of traditional Chinese medicines including Herba Kalimeridis Indicae、Cortex Phellodendri、Honeysuckle、Picrorhiza scrophulariiflora Pennel、Eugenia caryophyllata、Salvia miltiorrhiza Bge、Paeonia lactiflora Pall、Punica granatum L were extracted.The filter paper method was used to analyze the antibacterial activity of the extract and its active ingredients against Sau-06 multi-resistant Staphylococcus aureus,and the effective ingredient syringic acid of Herba Kalimeridis Indicae was determined to be an effective antibacterial drug.Furthermore,the minimum inhibitory concentration(MIC)and the minimum bactericidal concentration(MBC)of syringic acid inhibited Sau-06 multidrug-resistant Staphylococcus aureus were 0.8 mg/mL and 1.6 mg/mL,respectively,by the micro-double dilution method and the live bacteria counting method.The inhibitory effect of different concentrations of syringic acid on Sau-06 multidrug-resistant Staphylococcus aureus was dynamically displayed through the growth inhibition curve.The changes of intracellular ATP concentration,intracellular pH,and membrane potential were measured,it was found that syringic acid reduced intracellular ATP concentration and pH by destroying the integrity of the cell membrane,and triggered cell membrane hyperpolarization.At the same time,the results of transmission electron microscopy(TEM)and field emission scanning electron microscopy(FESEM)showed that 2MIC of syringic acid caused degradation of cell contents and changed of cell morphology.By FESEM and laser scanning confocal microscopy(CLSM),2MIC syringic acid had a good inhibit effect on biofilm formation of Sau-06 multidrug-resistant Staphylococcus aureus.RNA sequencing was conducted to determine the molecular mechanism of syringic acid inhibiting Sau-06 multi-resistant Staphylococcus aureus.Compared with the control group,a total of 592 genes had significantly different expressions.Among them,the expression levels of 266 genes were significantly down-regulated,and the expression levels of 326 genes were significantly up-regulated.In addition,the expression of sdrC,sdrD and clfB biofilm-associated genes were down-regulated,which proved that syringic acid inhibited the biofilm activity of the tested strains from the level of gene expression.Syringic acid affected arginine biosynthesis,the citrate cycle(TCA cycle),glycolysis/gluconeogenesis,nitrogen metabolism,degradation of aromatic compounds,and other pathways.This study illustrated the antibacterial activity of syringic acid against multidrug-resistant Staphylococcus aureus.In addition,Field emission scanning electron microscopy,Laser scanning confocal microscopy and other methods were used to analyze the mechanism of syringic acid in inhibiting multidrug-resistant Staphylococcus aureus.Finally,the molecular mechanism of antibacterial activity was analyzed at the level of transcriptomics.These results provided new candidate active ingredients for the treatment of multi drug-resistant Staphylococcus aureus infections.At the same time,it provides a certain theoretical basis for the clinical development of syringic acid.