Influence Of Echinacea Extract To Multi-drug Resistant Staphylococcus Aureus Biofilm Formation And Mouse Mastitis

Staphylococcus aureus(S.aureus)is prone to cause chronic and recessive intramammary infections(IMI)in dairy cows.The ability to form biofilm(BF)is a key factor in the development of IMI.BF is an organized,complex and fixed microbial community that can form on biological and non-living surfaces.The formation of BF can enable S.aureus to survive and persist in harsh environments.In recent years,due to the unreasonable clinical use of antibacterial drugs,the emergence of multi-drug resistant Staphylococcus aureus(MDR S.aureus)has led to the problem of difficult choice of therapeutic drugs.Echinacea purpurea(L.)Moench has dual effects of immune regulation and anti-inflammatory.Studies have shown that many plant active ingredients can effectively inhibit the formation of S.aureus BF.Therefore,this study explored influence of echinacea extract to MDR S.aureus BF and mouse mastitis,laying a research foundation for the clinical treatment of dairy cow mastitis by echinacea extract.In this study,the antibacterial effect of the combined application of echinacea extract and antibacterial drugs was determined by the checkerboard method;the inhibitory effect of echinacea extract alone and in combination with antibacterial drugs on the phenotype of BF formation in MDR S.aureus was determined by crystal violet staining method;the effect of different concentrations of echinacea extract on the BF formation of MDR S.aureus was determined by scanning electron microscopy;the effect of echinacea extract on BF formation related gene mRNA relative expression of MDR S.aureus was determined by RT-PCR;the pathological changes of breast tissues in different groups were observed by HE staining;the changes of ZO-1 and TLR-2 in breast tissues of different groups were observed by immunohistochemical section staining;the relative IL-1? mRNA changes in expression in breast tissues of different groups was determined by RT-PCR.The results are as follows:1.Echinacea extract alone and in combination with antibacterial drugs can significantly inhibit the BF formation of MDR S.aureus(P<0.01);the adhesion ability of MDR S.aureus continues to weaken with the increase of echinacea extract concentration,and the bacterial cells were deformed and swollen,and there was a dose-dependent effect;The genes related to the BF formation in MDR S.aureus were ica A,ica D,clf B and fnb B,after the effect of echinacea extract,the relative expression of ica D mRNA of MDR S.aureus was extremely significantly down-regulated(P<0.01),and the relative expression of clf B and fnb B mRNA was significantly down-regulated(P<0.05),the relative expression of ica A mRNA did not change significantly(P>0.05).2.Compared with the uninfected MDR S.aureus group,the boundaries of the acinar wall in the breast tissue of the infected MDR S.aureus group were not clear,the acinar epithelial cells were scattered and shedding,the ZO-1 of the breast epithelial cells was weakly positive,the tight junctions between the acinar epithelial cells was decreased,the TLR-2 of macrophages in the breast stroma showed a strong positive reaction,the acinar was filled with inflammatory cells,mainly neutrophils and macrophages,the relative expression of IL-1?mRNA was extremely significant rise.In echinacea extract treatment group and cefquinoxime sulfate treatment group,the breast acinar wall was clearly demarcated,the acinar epithelial cells were neatly arranged,there was lightly stained milk in the acinar,and there was only a small amount of inflammatory cell infiltration in the acinar,and the lobular space was only a small amount of neutrophil infiltration.Both echinacea extract treatment group and the cefquinoxime sulfate treatment group showed a moderately positive response to ZO-1,the degree of tight junctions between acinar epithelial cells was the same as uninfected MDR S.aureus group that can effectively protect the integrity of the breast epithelial barrier.Echinacea extract treatment group can significantly reduce the relative expression of IL-1?mRNA(P<0.01),and weaken the inflammatory response in breast tissue.The above results showed that echinacea extract alone and in combination with antibacterial drugs can significantly inhibit the formation of MDR S.aureus BF(P<0.01),and is dose-dependent;echinacea extract can attenuate the MDR S.aureus cause inflammation of mouse mammary tissues,and effectively protected the integrity of the mammary acinar epithelial barrier.