Analysis Of Genetic Evolution And Pathogenic Characteristics Of Streptococcus Uberis In Part Of China

In this study,the whole genome framework of bacteria was sequenced and compared,and the bacterial minimun inhibitory concentration（MIC）test was finished,as well as the drug resistance genes,virulence genes and drug resistance phenotypes of 38 S.uberis strains were obtained,then the sequence type（ST）and cloning complex（CC）of 38 S.uberis strains were analyzed by Mult-ilocus Sequence Typing（MLST）;and in order to analyze the genetic evolution relationship a phylogenetic tree were constructed;screen new ST strains for animal experiments to explore the differential immune response induced by S.uberis carrying different virulence genes;This is the first time to explore whether and how intestinal（flora）immunity plays a role in S.uberis mastitis.The purpose is to provide new ideas and methods for the prevention and control of S.uberis and similar mastitis pathogens and early diagnosis of mastitis.The research contents and results are as follows:1.Genetic evolution characteristics of S.uberis in parts of ChinaIn this study,38 strains of S.uberis isolated from some regions of China（mainly in Jiangsu and Xinjiang province）between 2010 and 2020 were used as the research object.Through the determination of MIC and sequencing of the whole genome framework,resistance and virulence genes and other relevant informations concerning 38 strains of S.uberis were obtained,then the above information is statistically distributed in time and space;based on MLST analysis of 7 housekeeping gene allele profiles,ST types and CCs;then explore whether there existed relationships among the dominant ST type and virulence and resistance genes;S.uberis,which is popular in Europe and America,and 38 S.uberis in this study were selected to establish a phylogenetic tree and analyze the genetic evolution relationship.The results showed that among the 15 antibiotics in the MIC test,except for ceftiophor and florfenicol,38 strains of S.uberis were resistant to 13 antibiotics to varying degrees.The resistance rate in 2020（Jiangsu province）was significantly higher than that in2010（Xinjiang province）;after the comparison of resistance and virulence genes,only 24strains of S.uberis carried the resistance gene,and the remaining 14 strains did not carry the resistance gene and originated in Xinjiang province,which was consistent with the results of the MIC test;15 virulence among the genes,with the exception of cfu,has A/B and lbp virulence genes,the remaining 12 virulence genes carried 100%.In the MLST results,38strains of S.uberis included 17 ST types,of which 15 were new ST types.Among them,ST968,ST1273 and ST1277 belonged to the dominant ST type,characteristically having and missing some resistance and virulence genes,but only 8 strains of S.uberis belong to ST-5 CCs.The results of the phylogenetic tree showed that the 38 strains of S.uberis in this time are far apart and the distribution is regionally dependent,and is quite different from the S.uberis prevalent in Europe and America.2.Pathogenicity analysis of new ST strain of S.uberisIn order to explore the pathogenic characteristics of the new ST type S.uberis and the host’s differential immune response,this experiment used three strains of S.uberis(SU41_ST1276,supan03_ST1281,HFJ5_ST986)and C57BL/6 mice as the research objects.Female rats were perfused with 3 strains of S.uberis（SU41 group,supan03 group,HFJ5 group）or corresponding volume of sterile normal saline（Control group）with mammary galnd catheter needle after delivery 48 h,then mammary galnd,liver,spleen tissues and plasma were collected 24 h later,were tested for related indicators subsequently.The results showed that the supan03 strain could induce more serious inflammation in the mammary galnd compared with the other two groups.However,the N-acetyl-β-D-glucosaminidase（NAGase）activity did not differ,compared with the Control group,the levels of MDA in plasma and liver tissues in the SU41 group increased significantly.Among the experimental groups in the mammary galnd,the total antioxidant capacity（T-AOC）of the supan03 group decreased significantly and the MDA level increased significantly（P<0.05）.At the genetic level,there was no difference in serum amyloid（SAA）in the liver and SAA and C Reaction Protein（CRP）in the mammary galnd among the experimental groups,while the m RNA expression of Tumor Necrosis Factor-α（TNF-α）in the liver in the supan03 group,TNF-α,Interleukin-6（Interleukin-6,IL-6）,Interleukin-1β（Interleukin-1β,IL-1β）and chemotaxis Factor 5（CC motif ligand 5,CCL5）in the mammary galnd were significantly higher than that of the Control group and the SU41 group（P<0.05）;the gene expression changes of high mobility group protein B1（HMGB1）and Toll-like receptor 4（TLR4）in the mammary galnd among the experimental groups were the same,supan03 group and HFJ5 group were significantly higher than SU41 group（P<0.05）,and the corresponding downstream NF-κB（p65）protein expression level is also significantly higher than the control Group（P<0.05）,indicating that strains carrying has A/B virulence genes can induce heavier tissue damage through the HMGB1/TLR4/NF-κB pathway.The above results indicate that infection with SU41 strain may cause temporary early immunity of the host,and then mainly maintain immunity,allowing the strain to exist for a long time;after infection with supan03 and HFJ5 strains,it will trigger the host’s strong immune defense function and induce stronger immune clearance.,Causing heavier tissue damage,among which the has A/B and lbp virulence genes have a synergistic effect to a certain extent.3.Preliminary screening of biomarkers for early diagnosis of S.uberis mastitisIn order to explore the intestinal（flora）functions after S.uberis infection,and whether there existed certain significant flora as early diagnostic biomarkers.In this study,the contents of the cecum of mice were collected aseptically,and 16S r DNA sequencing was performed.The results showed that there was no significant difference in the alpha diversity of the intestinal flora in each group;through principal coordinates analysis（PCoA）,non-multidimensional scaling（NMDS）and clustering analysis（UPGMA）)showed that the beta diversity of the intestinal flora after infection with S.uberis changed significantly（P<0.05）,but it might not be related to the ST type and virulence genes of the strain.When analyzing the composition of the intestine,the abundance of Verrucomicrobia and Firmicutes increased,the abundance of Proteobacteria and Actinobacteria decreased at phylum.At the genus level the abundance of Akkermansia increased significantly and the trend was in contrast with the above-mentioned damage degree of mammary gland.To based on the Bug Base and PICRUSt2 database to predict the bacterial phenotype and function,it was found that the ability of the intestinal flora to resist oxidative stress increased after S.uberis infection,and tended to form more short chain fatty acids（SCAFs）;LEfSe analysis showed that Akkermansia could be used as a specific biomarker after S.uberis infection.The above results indicated that Akkermansia played an important role in immune regulation after S.uberis infection and could be used as a biomarker for early diagnosis.To sum up,S.uberis in part of China were highly heterogeneous,and different ST types induced different host’s immune responses.Among them,Akkermansia in the intestine may play an important in immune response,it can be used as a biomarker for early diagnosis of S.uberis mastitis to a certain extent.The development of the research provided theoretical basis for the prevention and control of S.uberis and early warning of its risk of disease.