| With the release and implementation of the antibacterial ban policy in China,the selection and promotion of probiotics is an important fundamental work as the prevention and treatment of intestinal diseases and biofeed development.A lot of research has been done on the selection and development of Enterococcus durans.In addition,the preliminary team found that the addition of oregano essential oil to feed can effectively lead to a significant enrichment of Enterococcus durans(E.durans)strains in the intestine of sheep,which was significantly negatively correlated with the gene expression of pro-inflammatory factors and positively correlated with the gene expression of intestinal barrier protein.Comprehensive analysis of the previous studies showed that E.durans could be selected,identified,purified and cultured as a potential anti-inflammatory strain,and it could be studied and applied as a potential functional probiotic.In this study,E.durans 98 D was obtained by isolation and culture in the hindgut of healthy goats,and the probiotic properties and safety of this strain were analyzed by morphological identification,species identification,as well as physiological characteristics and whole genome analysis.By constructing a mouse model of colonic inflammation,the therapeutic mechanism of E.durans 98 D strain on intestinal inflammation and its effect on inflammatory intestinal flora were investigated.The results obtained from the experiment were as follows:(1)The strain E.durans 98 D was obtained from healthy goat feces with tolerance to hydrochloric acid and artificial gastrointestinal fluid,but not to bile salts and high temperature.In addition,the bacterium was able to protein-degrading enzyme activity.In addition,the self-agglutination and surface hydrophobicity of E.durans 98 D were 66.90 ±0.72% and 39.14 ± 2.02%,respectively,indicating that the strain has a high adherence and agglutination ability,which helps E.durans 98 D colonize the hindgut.strain E.durans 98 D did not exhibit hemolysis,and the culture supernatant of the strain was highly resistant to Escherichia coli(E.coli)Streptococcus pneumoniae(S.spneumoniae)Staphylococcus aureus(S.aureus)was significantly inhibited by the culture supernatant of the strain(P <0.01).The strains were highly sensitive to 18 antibiotics(62.07%),moderately sensitive to 5antibiotics(17.24%)and resistant to 6 antibiotics(20.69%).(2)The E.durans genome size is 2,983,334 bp with 38.05% GC content;the growth of coding genes is 2,487,903 bp and contains 2,718 coding genes,accounting for 83.39% of the genome.The E.durans genome in KEGG annotation contains NOD-like receptor pathways that regulate immunity and inflammation and encodes anti-folate related genes that play a role in the treatment of cancer and inflammatory diseases.In addition,the whole genome of E.durans contains few drug resistance genes;virulence factors are abundant,but mostly related to defense.Virulence factors are involved in anti-phagocytosis,adhesion,biofilm-produced sugar-binding transcriptional regulators,stress proteins,resistance to acute toxicity of bile and bile salts,etc.No pathogenic virulence factors were found.Therefore,durable enterococci have a good safety profile at the whole genome sequencing level,but the presence of drug resistance genes requires further evaluation of their safety at a subsequent stage.(3)E.durans 98 D treatment significantly reduced the pathological symptoms of intestinal inflammation induced by DSS,possibly by modulating the host immune response,decreasing the expression levels of pro-inflammatory factors TNF-α,IL-1β,and IL-6 genes(P < 0.05),elevating IL-10 anti-inflammatory factor gene expression(P < 0.05),and reducing immune factor infiltration in intestinal tissues(P < 0.05).Increased intestinal epithelial tight link protein gene expression(P < 0.05),restored intestinal barrier function,etc.alleviated intestinal inflammation in mice.In addition,E.durans 98 D treatment could reshape the intestinal microbiota of mice with colitis so that its composition converged to that of the control group to alleviate intestinal inflammation,increasing the abundance of Bacteroidota and Actinobacteriota flora at the portal level(P < 0.05)and Paraprevotella and Odoribacter genera increased in abundance(P < 0.05).In conclusion,E.durans 98 D can be exploited as an effective anti-inflammatory probiotic strain,which does not carry pathogenic virulence factors in the whole genome and has a high safety profile,but efrA and aac6-I resistance genes are present in the genome;therefore,the safety of the cut strain needs to be assessed by subsequent in vivo animal tests. |
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