The Study Of Inhibition Mechanism Of Tea Saponin On Streptococcus Agalactiae Biofilm From Dairy Cows

At this stage,the primary method for treating mastitis in dairy cows catused by S.agalactiae is still the injection of antibiotics.However,with the abuse of antibiotics,the biofilm of S.agalactiae has gradually become resistant to antibiotics.Therefore,new ideas for seeking other drugs to replace antibiotics or combining other drugs with antibiotics to treat biofilm infection have gradually attracted the attention of relevant scholars.Tea saponin extracted f rom Camellia plants is of great scientific interest with inexpensive,widely available,and few toxic side effects,whereas there are few reports on the inhibition of the Streptococcus agalactiae biofilm formation mechanism by tea saponin.This paper has been studied as an entry point.The experimental methods and results are as follows:1.The capsular polysaccharide serotype was identified for the isolates of S.agalactiae from dairy cows,and the biofilm formation conditions of S.agalactiae in vitro were optimized.The results showed that the S.agalactiae serotype was type Ⅲ;and in the TSB medium,the culture was formed for 48 h,the glucose concentration and the NaCI concentration were both 1.0%,the pH was 7.0,and the milk concentration was 1.5%.2.To study the effect of tea saponin on the biofilm formation of Streptococcus agalactiae,the growth curve experiment was used to test the effect of tea saponin on the growth of Streptococcus agalactiae strain.The biofilm of tea saponin was observed by scanning electron microscopy(SEM).The conclusions are as follows:With the increase of tea saponin concentration,the biofilm was inhibited gradually significantly;tea saponin had no effect on the total number of colonies during stable phase;the results of SEM showed that the experimental group was smaller,fewer,and sparser.The conclusion is that tea saponin can significantly inhibit the formation of biofilm.3.In this study,three strains(S.agalactiae,Escherichia coli,and Staphylococcus aureus)isolated from the same diseased cow were co-cultured to form a composite flora and the effect of tea saponin on the biofilm of the composite flora was studied.The effect of tea saponin on the composite biofilm was observed by SEM.The results showed that the composite bacteria could form a dense biofilm under low tea saponin concentration;while with the increase of tea saponin concentration,the resistance of the composite flora to tea saponin gradually decreased,similar with the results of SEM observations.The effect is that the high concentration of tea saponin can also inhibit the biofilm formed significantly by the complex flora.4.Amplifying the genes related to S.agalactiae biofilm by PCR technique,then detecting the transcriptional effects of tea saponin on biofilm-related genes by quantitative transcriptional PCR assay,there was no significant change in the transeriptional levels of the luxS and neuA genes,while the transcription levels of cpsE and·srtA were down-regulated by 73%and 50%,respectively.Moreover,as a biofilm negative regulatory gene,csrR was up-regulated by 37.8%.5.To test the effect of tea saponin on the release of eDNA from S.agalactiae biofilm,and the effect of tea saponin on the extracellular polysaccharide of S.agalactiae biofilm was detected by sulfonate method.The results showed that tea saponin inhibited the synthesis of water-soluble and water-insoluble extracellular polysaccharides.the effect of tea saponin on the release of S.agalactiae eDNA increased with the increase of its concentration,and the inhibition of eDNA trend showd a concentration-dependent effect.