| The widespread use and abuse of antibiotics in animal breeding has led to the emergence of drug-resistant pathogens,and antibiotic residues also pose a great threat to human health.Therefore,there is an urgent need to find new antibiotic substitutes for the prevention or treatment of bacterial diseases.In the process of animal breeding,Escherichia coli disease and Staphylococcus aureus infection are serious threats to the development of aquaculture,and even endanger human health and safety,which have been widely concerned because of their public health significance.Therefore,there is an urgent need to find effective treatments for this disease.Enterococcus has been used as a probiotic food additive or recommended as a supplement for the treatment of intestinal microbial disorders and other diseases.It can stimulate the body’s immune system,maintain the stability of the intestinal environment,synthesize vitamins and other important metabolites,and prevent the combination and spread of sporesome bacteria.In addition,Enterococcus can also play beneficial roles in cheese fermentation as well as in cheese ripening and development of specific flavor,texture and taste through proteolysis,esterification and lipolysis,citrate breakdown and production of diacetyl and other important volatile compounds.In this study,we screened and identified a group of probiotics with good antibacterial activity based on the laboratory,and obtained a strain of Enterococcus faecium with better antibacterial characteristics by comparing its antibacterial activity.We also evaluated its safety and studied its antibacterial mechanism,aiming to find Enterococcus with safety and potential application value,and to provide new materials for the development of Enterococcus related products.The main research contents and results of this thesis are as follows:1.Comparative screening of antibacterial activity probiotics and safety evaluation of Enterococcus faecium A7In order to further obtain the best bacteriostatic activity of probiotics,this study was based on five strains of probiotics with good bacteriostatic activity(Enterococcus faecium A7,D7,Tβ,Lactobacillus B2,Lactobacillus reuteri S5)screened in our laboratory previously.AGAR diffusion and biofilm tests were used to test the effects of five strains of probiotics on the viability and antibacterial activity of S.aureus S51 and E.coli AE17.The results showed that compared with Enterococcus faecium D7,Tβ,Lactobacillus B2 and L.reuteri S5,Enterococcus faecium A7 significantly inhibited the growth and biofilm formation(P<0.001)of S.aureus S51 and E.coli AE17.The safety of A7 was evaluated by virulence gene detection,hemolysis analysis and mouse safety test.The results showed that A7 did not contain virulence genes such as esq,hyl,ace,efa A and asal,and had no hemolysis on sheep blood AGAR plate,did not affect the mental state and growth of mice,and had no colonization in mouse organs.A strain of Enterococcus faecium A7 with high safety and good antibacterial activity was obtained,which laid a foundation for further research.2.In vitro antibacterial study of Enterococcus faecium A7 against Staphylococcus aureus and porcine E.coliIn order to further understand the antibacterial mechanism of Enterococcus faecium A7,cell-free fermentation supernatant(CFS)of A7 was extracted and its effective antibacterial components were analyzed.The results showed that the active substances of CFS were tolerant to temperature,catalase and proteinase K,but sensitive to p H,so the active substances may be organic acids or acid-dependent substances.The inhibitory effect of Enterococcus faecium A7 CFS on the growth of gram positive S.aureus and gram negative E.coli was observed.A7 CFS could cause shrinkage of S.aureus cell wall,damage of E.coli cell membrane and increase the permeability of cell membrane.Cell experiments showed that A7 could reduce the adhesion of pathogens to porcine intestinal epithelial cells.In summary,Enterococcus faecium A7 inhibits the growth of pathogens by secreting organic acids or acid-dependent substances,damaging the cell wall and membrane of pathogens,and adhering to host cells to resist the adhesion of pathogens.3.To analyze the antagonistic effect of Enterococcus faecium A7 against S.aureus S51 based on in vivo experimentsFirst,a mouse model of S.aureus infection was established,and the organ lesions of the mice were observed and the bacterial load in the organs of the mice was detected.The results showed that there were obvious bleeding spots in the lung and liver of the mice,but no obvious lesions in the heart,spleen and kidney.S.aureus was detected in the heart,liver,spleen,lung and kidney,and the number of S.aureus in the liver and lung was higher.This indicated that the mouse infection model was successfully constructed.KM mice were divided into blank control group,S.aureus S51 infection group,A7+S51 prevention group,and S51+A7 treatment group.The mice in the blank control group were given sterilized normal saline,the mice in the S.aureus group were given S.aureus for 7 consecutive days,and the mice in the prevention group were given Enterococcus faecium A7 for 7 consecutive days followed by S.aureus for 7 consecutive days.The treatment group was treated with Enterococcus faecium A7 by intragastric administration followed by S.aureus for 7 days.After the end of challenge,the mice were sacrificed on the 7th day after challenge for histopathological examination,and the expression of inflammatory factors and intestinal proteins was detected by fluorescence quantitative PCR.The results showed that the A7+S51prevention group could alleviate the decrease in the specific weight of the liver caused by S.aureus invasion,effectively alleviate the liver and lung injuries and the rupture of the small intestinal villi caused by S.aureus invasion,and reduce the expression of inflammatory factors TNF-α,IL-6,IL-1β and IL-8(P < 0.001).Increasing the expression of small intestinal lysozyme LYZ-2 can effectively alleviate the invasion of S.aureus.A7 treatment also effectively alleviated the damage of liver and lung and intestinal villi caused by S.aureus infection,but it could not antagonized the increased expression of inflammatory factors in liver and spleen caused by S51 infection.In conclusion,this study successfully obtained a probiotic strain of Enterococcus faecium A7,which can inhibit the activity of S.aureus and E.coli and inhibit the adhesion of pathogens to host cells by destroying the bacterial cell membrane by producing organic acids.In vivo experiments showed that Enterococcus faecium A7 had a good preventive effect against S51 infection.This study can provide certain data and theoretical basis for probiotics in the prevention of E.coli and S.aureus infections in clinical practice. |
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