Study On The Mechanisms Of Buqiyin Formula Against MRSA Infection Based On C-di-AMP Signal Pathway

Background:Multi drug resistant bacterial infections are a major threat to human life and health today.Methicillin resistant Staphylococcus aureus(MRSA)is an important pathogenic bacterium that can cause multi-system infections.The high pathogenicity,recurrence rate,and mortality rate of MRSA are closely related to its high resistance to various clinical antibiotics.The formation of biofilm(BF)is an important reason for its drug resistance.Zhenqi granules(ZQ)exerts antibacterial and enhancing immunity activities in clinical practices,and can be used to combat MRSA infection.Objective:To determine the effect of ZQ combined with vancomycin(Van)against planktonic MRSA and MRSA with BF(MRSA-BF),and preliminarily to reveal its synergistic antibacterial mechanisms.To find the protective effect of ZQ on BEAS-2B pulmonary epithelial cells infected with MRSA.Methods:The minimum inhibitory concentration(MIC)and minimum bactericidal concentration(MBC)of Van and ZQ against MRSA was detected by microdilution method and plate marking method.The fractional inhibitory concentration index(FICI)of MRSA was detected by chessboard method to determine the MIC ratio.Van and ZQ were used alone and in combination to intervene in MRSA-BF.The changes in the number of live bacteria in MRSA-BF were detected by microplate method.The changes in the number of live and dead bacteria in MRSA-BF were observed by confocal laser confocal microscopy(CLSM).The effects of Van and ZQ alone and in combination on the morphology of MRSA-BF were observed by scanning electron microscopy(SEM).RT-PCR was used to detect the effects of Van and ZQ alone and in combination on the expressions of MRSA-BF related genes purF,lytS,IrgB,tarH,dltA,fmtA,and krtA.ELISA was used to detect the effects of Van and ZQ alone and in combination on the expression of c-di-AMP signaling molecules.The optimal concentration of ZQ intervention in BEAS-2B cells was determined by CCK8;The effect of ZQ on the adhesion ability of MRS A and BEAS-2B cells was observed through co-culture models and adhesion experiments.Results:The MIC for Van was 2 μg/mL.The MIC of ZQ cannot be detected,so 800 mg/mL,the maximum solubility of ZQ,was determined as the experimental concentration.The FICI value of the two drugs was not detected in the MIC ratio experiment.200 mg/mL ZQ has a significant inhibition effect on MRSA-BF membrane live bacteria.Morphological observation found that there were no differences in the fluorescence signal intensity of live and dead bacteria between the 1 μg/mL Van group and the Control group.The fluorescence signal range of live bacteria in 200 mg/mL ZQ group was less than 1 μg/mL Van group and Control group.The fluorescence intensity of live and dead bacteria in the 1μg/mL Van+200 mg/mL ZQ group was lower than that in the Control group and those drugs alone groups.The effect of 1 μg/mL Van on the morphology of MRSA-BF was no difference from that of the Control group.In the 200 mg/mL ZQ group,there was no membrane structure coating on MRSA,only some micro-colonies were scattered and arranged loosely among the cells.In the 1μg/mL Van+200 mg/mL ZQ group,MRSA had no membrane structure coating,and the arrangement between the bacteria was loose.Compared with Control group,the expressions of seven related genes,purF,lytS,IrgB,tarH,dltA,fmtA,and krtA were inhibited in the combination intervention of two drugs.The protein of c-di-AMP,pathway signaling molecule,was also inhibited.The optimal concentration of ZQ on BEAS-2B cells was 12.5 mg/mL,and when the infection ratio(MOI)was 1:1,10mg/mL and 12.5mg/mL ZQ can significantly inhibit the adhesion of MRSA to BEAS-2B cells.Conclusions:1.ZQ has an inhibitory effect on MRSA-BF,which can assist Van in contacting bacteria in BF,and promote Van’s clearance of MRSA.2.The mechanism of Van combined with ZQ in inhibiting MRSA-BF is to inhibit the expression of genes such as fmtA and dltA,inhibiting BF production,destroying cell wall structures,and reduce the release of extracellular DNA by reducing bacterial autolysis.3.ZQ has an inhibitory effect on the adhesion ability of MRS A,which can inhibit the adhesion of MRSA to BEAS-2B cells,thereby reducing the colonization of MRSA on the cell surface,and protecting BEAS-2B cells.4.This study provides theoretical basis for the clinical treatment of MRSA with ZQ combined with Van,and lays a foundation for the development of Traditional Chinese Medicine combined with antibiotics in the treatment of refractory drugresistant bacterial infections.