Study On The In Vitro Antibacterial Activity Of Andrographolide Sulfonated Against MRSA Based On Biofilm

Background: bacterial biofilm(Biofilm,BF)is an important cause of chronic and severe bacterial infectious diseases.Bacteria that form BF are more resistant to antibiotics than suspended bacteria.Methicillin-resistant Staphylococcus aureus(Methicillin-resistant Staphylococcus aureus,MRSA)uses the formation of BF to hinder the killing effect of antibiotics,which is one of the main obstacles to the treatment of infection.Therefore,the inhibition of bacterial BF is very important for antibiotics to play an antibacterial role.Andrographolide sulfonated(Andrographolide sulfonated,AS)is a natural bioactive component,which has attracted much attention because of its extensive pharmacological activities.It is an effective component of andrographolide sulfonate extracted from andrographis paniculata.It has antiviral,anti-inflammatory,antibacterial,immunomodulatory and other pharmacological activities.However,its anti-BF mechanism is not clear.The purpose of this study was to explore the inhibitory effect of AS on MRSA and its biofilm,and to explore the mechanism,so as to provide a new method for the clinical treatment of MRSA and its biofilm.Methods: the minimal inhibitory concentration(MIC)of AS was determined by microdilution method,the effect of different concentrations of AS on the growth of MRSA for 24 hours was determined by growth curve method,the effect of AS on the number of living bacteria in mature MRSA-BF was determined by XTT reduction assay,and the morphological characteristics of mature MRSA-BF were observed by scanning electron microscope(SEM)and laser confocal scanning microscope(CLSM).In addition,the permeation effect of AS on MRSA-BF was detected by BF permeation test;Real Time PCR was used to detect the effect of AS intervention on the mRNA expression of genes related to BF formation,such as virulence factor sarA,regulatory gene cidA and quorum sensing system(quorum sensing,QS)related gene luxS.Results: the MIC of AS on MRSA measured by microdilution method was 50 mg / ml.The growth curve analysis showed that AS had a certain inhibitory effect on the growth of MRSA for 24 hours,and the inhibitory effect showed a certain dose-dependent relationship.The results of XTT reduction assay showed that AS could significantly inhibit the formation of MRSA-BF in the concentration range of 0.39-50mg/ml,and AS had a very significant inhibitory effect on the formation of MRSA-BF in the concentration range of 3.125-50mg/ml.CLSM observation showed that after the treatment of AS,the thickness of mature MRSA-BF decreased,the fluorescence signal of living bacteria weakened,the fluorescence signal intensity of dead bacteria increased,and the production of extracellular matrix was inhibited.The results of scanning electron microscope showed that the structure of BF disappeared,the cell size was different,the cell was scattered,and the cell division was abnormal.The results of BF permeation test showed that the structure of BF was destroyed after the intervention of AS,so that antibiotics could pass through the filter membrane effectively,thus exerting antibacterial effect.The results of RT-PCR detection showed that AS could significantly down-regulate the mRNA expression of MRSA-BF-related virulence factor SarA,regulatory factor cidA and QS system-related gene luxS at the concentration of 1/2MIC,and the inhibitory effect of luxS and cidA of AS at 1/2MIC concentration was more significant than that of Van group.Conclusion: AS can inhibit MRSA and its BF.AS acts as an anti-biofilm by inhibiting virulence factors,regulatory factors and QS systems related to the formation of MRSA-BF.AS can significantly inhibit the formation of MRSA-BF the main mechanism is to inhibit the expression of genes such as SarA,regulator cidA related to MRSA-BF formation and luxS related to QS system and interfere with the synthesis of extracellular matrix.