Effects Of Matrine On Virulence And Virulence Genes Of Streptococcus Agalactiae Isolates From Milk And Its Mechanism

Streptococcus agalctiae(S.agalactiae),also known as Group B Streptococcus(GBS),is currently one of the main pathogens that cause mammary gland inflammation in dairy farms in China,because GBS mostly does not cause mastitis With clinical symptoms,many farmers cannot detect the infection,which has caused significant losses to the dairy farming industry.The milk quality and milk production of dairy cows are directly related to the health of the mammary glands.Therefore,ensuring the health of the mammary glands of dairy cows is a necessary guarantee for the production of high-quality milk.Natural plant extracts are unique in the treatment and prevention of mastitis due to their relatively low price and good therapeutic effect.In recent years,studies have found that matrine not only has antioxidant,immunomodulatory,and protective effects on cardiovascular endothelial cells,but it can also regulate the expression of related inflammatory factors,regulate apoptosis-related pathways,and inhibit the heart Muscle fibrosis and other methods protect the blood circulatory system,and can prevent the occurrence of cancer.Therefore,the application and promotion of matrine in animal husbandry production has broad prospects under the background of completely banning antibiotics.This study aimed to investigate the effect of matrine on the virulence of Streptococcus agalactiae.The test was conducted in September 2020 from a commercial dairy farm in Beijing using the California Mastitis Rapid Test Reagent(CMT)to conduct a preliminary screening of dairy cows.The CMT result was "+" or higher and the somatic cell(SCC)test result was 200,000/m L or more.Cows with recessive mastitis undergo further testing.The pathogens in the milk are isolated and cultured,and the species is identified by Gram staining,biochemical tests,and bacterial 16 S r RNA gene sequencing and clone sequencing.Different concentrations of matrine were co-cultured with the standard bovine strain(ATCC 13813)and the clinically isolated strain GBS,and the changes in the MIC and growth curve were measured.And through whole blood killing test,adhesion test and q RT-PCR methods,explore the effect of matrine on the expression of related virulence genes of Streptococcus agalactiae.The results of the study showed that after more than 600 lactating cows were combined with CMT detection,SCC analysis and 16 S sequencing results to identify the isolated strains,a total of 3 clinically isolated Streptococcus agalactiae(GBS1,2 and 3)were obtained,and they were gram as a positive bacteria,a single cell is spherical and multiple cells are arranged in chains.The biochemical test results show that the contact enzyme test of Streptococcus agalactiae is negative and the CAMP test is positive.Matrine was co-cultured with the standard strain for 24 hours,and at 2 mg/m L,there was a very significant difference from the untreated group(P<0.01),and when the concentration was higher than 4mg/m L,it completely inhibited the growth of bacteria(P <0.01).From the growth curve results,it can be seen that Streptococcus agalactiae enters the logarithmic phase at 6 h;when matrine is at 2 mg/m L,it affects the growth of standard strains at 6 h,and enters the plateau phase at 12 h;at 4 mg/m L Completely inhibit the growth of standard strains.The results of clinically isolated strains show that the MIC of clinically isolated strains(12,10,and 8 mg/m L)is far from the standard strain(2 mg/m L).The results of the whole blood killing test show that matrine at 4 mg/m L can effectively promote the killing ability of blood against Streptococcus agalactiae,reduce the immune defense ability of Streptococcus agalactiae,and make the survival rate of Streptococcus agalactiae in the blood.Decrease to 70.40%(P<0.05);when matrine concentration is 6 mg/m L,its survival rate in the blood is reduced to 41.40%(P<0.05);when the drug concentration is 8 mg/m L,it is in The survival rate in the blood dropped to17.98%(P<0.05).Biofilm crystal violet staining results show that matrine can effectively inhibit the formation of Streptococcus agalactiae ATCC13813 biofilm at 2 mg/m L,and significantly inhibit the formation of a clinically isolated strain GBS1 biofilm with strong biofilm at 8 mg/m L The generation.The results of the adhesion test showed that when the concentration of matrine was 2 mg/m L,the adhesion rate of the standard strain to dairy cow mammary epithelial cells was reduced to 63.2%(P<0.05),and when the concentration of matrine was 6 mg/m L,The adhesion rate of clinically isolated strains to breast epithelial cells decreased to 14.6%(P<0.05).In addition,the results of transcriptomics showed that matrine adjusted a total of 650 genes,up-regulated 469 genes,and down-regulated 181 genes in the standard strain of Streptococcus agalactiae.The clinical isolates regulate 1046 genes,among which 645 genes are up-regulated and 401 genes are down-regulated.They are enriched in the three biological processes of biological processes,cell components,and molecular functions.Pathways related to virulence and significantly changed in both clinically isolated strains and standard strains are:glycosaminoglycan degradation,peptidoglycan biosynthesis,ABC transporter,two-component system,quorum sensing,biofilm formation,Bacterial chemotaxis,protein secretion and bacterial secretion system.These pathways are respectively related to capsule production,exotoxin secretion,virulence factor secretion and regulation,and biofilm formation.The results of q RT-PCR showed that the standard strain of 2 mg/m L,GBS1 10 mg/m L matrine was co-cultured with Streptococcus agalactiae for 24 h.GBS1 and ATCC13813 were associated with adhesion-related ? subunits of protein C(Bac),C protein? subunit(Bca),laminin binding protein(Lmb),fimbrin(PI-2b)and fibrinogen binding protein Fbs A gene expression decreased significantly(P<0.05);and The expression of hemolysin(Cyl E),CAMP factor(CAMP),hyaluronidase(Hyl B)and capsular polysaccharide(Cps A)related to invasion and immune escape was significantly decreased(P<0.05).The above results show that matrine can effectively inhibit the expression of genes related to the invasion and adhesion ability and immune evasion ability in the virulence factor of Streptococcus agalactiae,thereby reducing the adhesion to the cow's mammary epithelial cells and reducing the damage to the breast tissue,Maintain breast health.In summary,matrine has a significant effect on the regulation of the virulence of Streptococcus agalactiae,which provides a theoretical basis for matrine as an effective drug for the prevention and treatment of mastitis.