The Study On Mechanisms Of Bile Salts Resistance In Enterococcus Faecium

As a potential substitute of feed antibiotic additives,probiotics have attracted extensive attention due to its role in maintaining animal health and performance.The survival in the gastrointestinal tract when probiotics administered would directly influence its probiotic effects.The bile salts that secreted by the host liver play an important role in fat metabolism.At the same time,bile slat is one of the antimicrobial substances for gastrointestinal bacteria as well.Thus,the resistant ability to bile salts is essential for the survival and persistence of intestinal bacteria in gastrointestinal tracts.However,the molecular mechanism of bile salts resistance in probiotic is still unclear.To elucidate the molecular mechanism of probiotic bile salt tolerance is of great significance for the screening and application of probiotics.In addition,the genetic background of probiotics should be clarified and safety assessment should be strengthened when screening probiotics in order to prevent potential risks in practical applications.Enterococcus faecium is one of the probiotics for feeding and belongs to the normal residents in the chicken gastrointestinal tract,which can maintain intestinal health and improve performence.Therefore,in this study,we firstly screened and characterized E.faecium isolates from broiler gastrointestinal tracts,then assessment of their saftey were performend by comparative genomics.In addition,we also studied the mechanism of two-component system and bile salts hydrolase（BSH）in bile salts resistance in E.faecium.The main results were shown as follows:1.Eight isolates of E.faecium were screened out from 106 strains of lactic acid bacteria that were isolated from the gastrointestinal tract of healthy broilers by acid tolerance test and16s rDNA sequence species identification.Then,through assessing the abilities in bile salts tolerance,adhesion to epithelial cells,antimicrobial activity against pathogenic bacteria,hemolysis activity,biofilm formation,antimicrobial susceptibility and virulence genes,E.faecium NW2 was selected for further study,because its tolerence to acid and bile salts,highest adhesion ability to epithelial cells,and antimicrobial activity against pathogenic bacteria.At the same time,it is sensitive to all tested antibiotics,has no hemolysis ability,does not produce biofilm,and does not contain any of three virulence genes（IS16,esp and hyl）.The results showed that E.faecium NW2 has good probiotic characteristics and high safety,and is an ideal candidate for probiotics.2.The genetic background of E.faecium NW2 was revealed by whole genome sequencing.It was found that NW2 contains 2756 ORFs,63 tRNA genes and 4 rRNA.Comparative genomic analysis revealed that NW2 carries a total of 13 heavy metal resistance genes,two BSH genes,and four multidrug efflux pumps,which give NW2 better resistance to stress.The whole genome analysis revealed that NW2 contains two potential pathogenic genes,acm and efaA_fm,but nonsense mutation were found in CDS region that results in nonfunctional protein product of acm and efaAfm,so they have no pathogenic ability.Phylogenetic analysis with SNP revealed that NW2 is closely related to probiotic E.faecium WEFA23 and is far from clinical pathogenic strains DO and Aus004.In addition,compared with the genome of the NW8 isolate with low bile salt tolerance,it was found that there are different in the amino acid sequence of two-component system proteins between NW2 and NW8,which may be the cause of the difference in bile salt tolerance.These results further confirmed the safety of NW2.3.The mechanism of two-component systems in bile salts tolerance of E.faecium was investigated.The results showed that the gene expressions of 2 two-component systems,LiaFSR and BsrXRS,were significantly positively correlated with the survival rate of E.faecium exposure to bile salts.It was further confirmed that LiaRS and BsrRS can regulate the bile salts resistance of E.faecium by gene mutations of BsrRS and LiaRS.The regulatory functions of the BsrRS system have not been studied.It was found that BsrRS were directly autoregulated,and 27 BsrR potential downstream target genes were identified using multiple molecular approaches.The target gene functions involved in cell membrane fluidity,fatty acid biosynthesis,cell wall homeostasis,DNA mismatch repair.4.Molecular cloning,characterization and comparison of bile salt hydrolases from E.faecium.It was found that NW2 has hydrolysis ability to both glycine and taurine-conjugated bile salts by the plate screening method.Analysis of the genome revealed that NW2 has two bile salt hydrolases:BSH1 and BSH2 which share with 83%identity of amino acid sequence.Recombinant expression of two BSHs in E.coli showed that the substrate specificities of the two BSHs were similar,and they both had strong hydrolysis ability to glycine and taurine-conjugated bile salts bile salts,and had the strongest hydrolysis ability to taurodeoxycholic acid.The optimum temperature and pH of the two BSH activities were differen.The optimum temperature of BSH1 and BSH2 activities were 37^oC and 30^oC respectively,the optimum pH of BSH1 and BSH2 activities were 6.0 5.4,respectively.In addition,17 compounds that are used as dietary supplements in animal feeds were selected for testing their inhibitory effects on the activity of the BSH.It was found that CuSO4inhibited BSH1 activity and had little effect on the activity of BSH2;KCl,NaCl,NaI,KI,NaSO₄,MgSO₄,NaHCO₃ did not affect the activity of BSH1 and inhibited the activity of BSH2;MnCl₂,CoCl₂,CaCl₂,ZnSO₄,MnSO₄ and FeSO₄ inhibited BSH1 and BSH2;CuCl₂,ZnCl₂ and FeCl₃ had little effect on the activities of BSH1 and BSH2.In summary,this study combined the traditional probiotic screening methods with genome-wide sequencing and analysis to screen out a probiotic E.faecium NW2 with good probiotic characteristics,high safety and high bile resistant ability.In addition,we found that the LiaFSR and BsrXRS systems contribute to biles salts resistance in E.faecium.We also identified two BSH that involved in bile salt tolerance.These findings provide a theoretical basis for optimizing the screening application of probiotics and the development of efficient engineering probiotics.