Study On The Effect Of Brazilin On Biofilms Of Staphylococcus Aureus

Objective: To explore the inhibitory effect of brazilin(BN)on biofilm formation and to evaluate the damaging effect of BN with or without vancomycin(VCM)on the biofilm.Which would provide experimental basis for treatment biofilm relation affections of S.aureus.Methods: 1.Effect of BN on S.aureus biofilm formation: Firstly,the biofilm model of S.aureus was established,by Congo red screened clinical strains,which it could formate biofilm.Using Safranin staining method was developed for the determination of S.aureus biofilm formation ability.And then,by crystal violet staining observed the adhesion of biofilm,fluorescein isothiocyanate-conjugated concanavalin A(FITC-ConA)marked fluorescence microscopy and scanning electron microscopy(SEM)observed of biofilm formation and identified of the biofilm model.Secondly,the antimicrobial susceptibility testing of BN and VCM was performed using the standardized broth microdilution method.Lastly,VCM was used as a positive control.The biofilm treated with 1MIC,1/2 MIC,1/4MIC BN and 1MIC VCM for 6h,12 h,24h and 48 h to assessed whether BN could inhibit biofilm formation.We obtained OD values and images by crystal violet staining and fluorescence microscopy.2.Effect of BN combined with VCM damaged S.aureus biofilm: BN with or without VCM were added to each well for 6h,12 h,24h and 48 h.The concentrations of BN were 2 MIC,1 MIC,1/2 MIC and VCM were 2 MIC,1 MIC,1/2 MIC.Crystal violet staining was performed and reading values at OD570 nm and the biofilm bacterial CFU counts were conducted.The biofilms was treated BN with or without VCM for 48 h,we used fluorescence microscopy,confocal laser Scanning microscope(CLSM)and SEM to explore the effects of BN and VCM to the mature biofilm of S.aureus.3.Expression of genes forming biofilms as detected by qRT-PCR: Using Trizol method to extract RNA of SA biofilm and quantitative real-time polymerase chain reaction(qRT-PCR)detect the expression of intercellular adhesion A(icaA),intercellular adhesion regulatory R(icaR)and accessory gene regulator A(agrA),which the mature biofilm treated with 1/2MIC,1MIC and 2MIC for 6h,12 h,24h and 48 h.Results: 1.Effect of BN on S.aureus biofilm formation: 84% clinical strains could form biofilms in 50 clinical strains.we chosen C-4-4 because it has the strongest ability to form biofilms.By adhesion experiment,2 to 4h almost no bacterial adhesion onto a glass slide,4to 6 h with a few bacteria,6 to 18 h bacteria gathered each other,18 to 36 h could see purple patches,36 to 48 h increased thickening and purple patches,48 to 72 h bacteria become loose.Fluorescence microscopy observed S.aureus biofilm formation,6h there were a few scattered bacteria,less green fluorescence;12h bacteria gathered,green fluorescence increased;24h there were small block green fluorescence;48h dense and heavy green fluorescence;72h green become loose.SEM observation of biofilm formation,6h bacteria are scattered in the distribution;12h bacteria began to gather and a small amount of extracellular matrix;24h bacteria enclosed by extracellular matrix;48h you could see the membrane structure;72h bacteria become loose.The MICs of BN for the C-4-4 and ATCC25923 strains were 32 ?g/ml.The MIC of VCM for the two strains was 0.5 ?g/ml.The biofilm treated with 1MIC,1/2 MIC,1/4MIC BN for 6h,12 h,24h and 48 h,showed all of them had significant inhibitory effect on the formation of biofilms,in addition to 6h and12 h 1/4 MIC of BN.While the best effect was at the time of 24 and 48 h,which optical density value fell by more than 2 times,respectively from 0.726±0.119 to 0.346±0.127 and from 1.133±0.283 to 0.450±0.121.By fluorescence microscopy the effects of BN on biofilms formation increased with the concentration increasely.Which,at 48 h 1MIC,were only a small amount of thin dotted green fluorescence.2.Effect of BN combined with VCM damaged S.aureus biofilm: BN with or without VCM were added to each well for 6h,12 h,24h and 48 h,the results showed that the effect of the combination was better than alone.And the best effect was appeared with 2MIC BN +2MIC VCM for 48 h.OD values was obviously decreased from 1.283±0.272 to0.390±0.160.And the colony count was also obviously down from 256.333±13.204 to47.333±11.604.It showed that there were only a small amount of thin dotted green fluorescence under fluorescence microscopy,but a large number of red fluorescence under CLSM.By SEM the biofilms were damaged and a lot of individual bacteria.3.Gene expressing: The gene of biofilm treated with 1MIC,1/2 MIC,1/4MIC BN for 6h,12 h,24h and 48 h,the results showed that BN downregulated the gene expression of icaA.The best effect was appeared with 2MIC BN for 48 h,values was obviously decreased from1 to 0.313±0.917.And BN upregulated icaR and the quorum-sensing system regulatoragrA,values was obviously increased from 1 to 4.581±0.560 and 3.049±0.225 respectively.Conclusion: 1.BN inhibited biofilm formation in a dose-dependent.2.BN combination with VCM generally enhanced damage biofilms,while VCM alone did not.The destruct effect of BN on the SA biofilm and the synergistic bactericidal effect with VCM.3.BN could down-regulate the gene expression of icaA,up-egulated icaR and the quorum-sensing system regulators agrA,which are related to the biofilm formation.