Research Of Anti-infection Of Traditional Chinese Medicine Based On Biofilm Model In Vitro

Staphilococcus aureus(S.aureus)is the leading cause of hospital and community-acquired infections and poses a significant burden on the healthcare system.When S.aureus attaches to medical devices and host tissue,it can easily form biofilms and cause chronic infections.In addition,the formation of biofilms reduces the sensitivity of the bacteria to host defense systems and antibacterial drugs,which contributes to the continuous spread of bacteria in chronic infections.Traditional Chinese medicines(TCM)not only come from a wide range of sources,have little toxic and side effects,but also can eliminate or reverse bacterial drug resistance.Hence,they become a new hotspot in anti-biofilm infection.The purpose of this study is to establish a research method for the anti-biofilm activity of TCM components.The anti-biofilm activity was investigated by basing on biofilm model in vitro.And the effects on the formation of biofilm and the clearance of mature biofilm were studied from the aspects of quantitative analysis,activity detection and content changes of biofilm matrix components.The main research contents are as follows:1.Selection of antibacterial drugs.The effects of concentration,suspension concentration,medium type and reaction time on the resazurin method were investigated.The final reaction conditions were as follows:The medium was LB,and the final concentration in the well was 20μg/m L,the concentration of bacterial suspension was 5×10~5CFU/m L,and the reaction time was 9 h.The activities of rhein,plumbagin,gossypol,quercetin and berberine hydrochloride against S.aureus in vitro were determined by resazurin method and microbroth dilution method.The results showed that the minimum inhibitory concentration(MIC)of five TCM components measured by the two methods were consistent.The MIC of rhein,plumbagin,gossypol,quercetin and berberine hydrochloride were 8μg/m L,4μg/m L,1024μg/m L,256μg/m L and 64μg/m L,respectively.Among them,rhein and plumbagin had significant bacteriostatic effect,while gossypol required higher concentration.Resazurin method has high accuracy and sensitivity and is suitable for high-throughput screening of drugs against S.aureus.The minimum bactericidal concentration(MBC)was determined according to whether there was colony growth on nutrient agar.The MBC of rhein,plumbagin and berberine hydrochloride were 256μg/m L(32MIC),64μg/m L(16MIC)and 1024μg/m L(16MIC),respectively.When the concentration of quercetin was 1024μg/m L,there was still colony growth,so its MBC was>1024μg/m L.The effects of different concentrations of TCM components on the growth curve of S.aureus were further studied.It was found that when the concentration of rhein,plumbagin,quercetin and berberine hydrochloride was MIC,the bacterial growth was significantly inhibited,and the attenuation percentages were 90.46%,91.62%,85.85%and 90.99%,with the growth rate below 0.01.It showed that the growth of S.aureus was almost completely inhibited at the concentration.2.Construction of S.aureus biofilm model.S.aureus biofilm was studied in different suspension concentrations(1×10~8CFU/m L,1×10~7CFU/m L,5×10~6CFU/m L,1×10~6CFU/m L,5×10~5CFU/m L,1×10~5CFU/m L),cultivation environments(stationary culture,30 rpm),temperatures(23℃,37℃,48℃),medium types(LB,MH,TSB,M63)and glucose contents(1%,2%,3%,5%,10%).The results of crystal violet staining showed that in a certain range,the increase of bacterial suspension concentration could add the amount of biofilm formation.When the concentration of bacterial suspension was 1×10~6CFU/m L,the amount of biofilm formation reached the maximum,but the amount of biofilm formation decreased as the concentration of bacterial suspension continued increasing.Compared with 30 rpm,the resting state was more suitable for biofilm growth.The amount of biofilm formation at 37℃was significantly higher than at other temperature conditions.LB medium had the largest amount of biofilm formation,while M63 medium had almost no biofilm formation.With the increase of glucose content,the amount of biofilm formation climbed up and then declined.When glucose content was 3%,the amount of biofilm formation reached the highest,which increased by 32.6%compared with that without glucose.Therefore,the optimized culture model of biofilm were:With LB medium containing 3%glucose,S.aureus at a concentration of 1×10~6CFU/m L cultivated in 37℃for static culture.3.Biofilm inhibition activity analysis of TCM.Firstly,crystal violet staining method was used to measure the amount of biofilm formation,XTT method was used to measure the metabolic activity of biofilm.The biofilm growth curve is plotted by these two methods.After3 days of culture,the total amount of biofilm formation and the metabolic activity of bacteria in the biofilm reached the maximum,and mature biofilms were formed.Then,the inhibitory activities of rhein,plumbagin,quercetin and berberine hydrochloride on biofilm were discussed by studying the formation and removal of biofilm.The results were as follows:all the four TCM components had obvious inhibition effects on the formation of S.aureus biofilm.The total amount of biofilm formation decreased by more than 80%when the concentrations of rhein,plumbagin,quercetin and berberine hydrochloride were 8μg/m L,16μg/m L,32μg/m L and 64μg/m L,respectively.When the concentrations of rhein and plumbagin were 32μg/m L,the concentrations of quercetin and berberine hydrochloride were128μg/m L,the metabolic activity of bacteria decreased by more than 80%.The four TCM components also had certain cleanning effect on mature biofilms.The removal rate of mature biofilms was more than 80%when the concentrations of rhein,plumbagin,quercetin and berberine hydrochloride were 64μg/m L,256μg/m L,128μg/m L and 1024μg/m L,respectively.When the concentrations of rhein,plumbagin,quercetin and berberine hydrochloride were 256μg/m L,1024μg/m L,512μg/m L and 4096μg/m L,the metabolic activity of bacteria decreased by 82.21%,93.83%,85.41%and 78.68%.4.Influence of TCM monomers on the extracellular matrix components of biofilms.Firstly,we confirmed that polysaccharide,protein and DNA affected the amount of biofilm formation by the enzymolysis experiment,among which DNA had the most obvious effect.Congo red plates were used to determine the content of PIA.The results showed that the four monomers all had a certain inhibitory effect on the production of PIA,and the inhibitory effect increased with the augment of drug concentration.The results of extracellular protein by micro BCA showed that all the four components could inhibit the protein content in biofilm matrix.Among them,rhein and plumbagin had obvious inhibitory effect.The inhibition rates were 86.51%and 86.62%at 16μg/m L and 8μg/m L,respectively.In addition,rhein and plumbagin significantly reduced the release of eDNA in the biofilm matrix.In this thesis,the working conditions of the resazurin were optimized,which provided a new idea for the high throughput screening of TCM against Staphylococcus aureus.The model of biofilm in vitro was established by single factor experiments.The inhibitory activities of rhein,plumbagin,quercetin and berberine hydrochloride were determined based on the total amount of biofilm and the metabolic activity of bacteria in the biofilm.The effects of four traditional Chinese medicine components on the contents of PIA,protein and eDNA in the biofilm matrix were further studied.This research successfully established the method of biofilm inhibition activity of TCM components,which provided laboratory support for the development and utilization of anti-infective drugs.