Unlocking the power of swine gut bacteria: newly isolated Blautia strain and its metabolites inhibit the replication of Salmonella Typhimurium in macrophages and alleviate DSS-induced colitis in mice

Abstract

Background: Inflammatory bowel disease is a significant health concern for both humans and large-scale farm animals. In the quest for effective alternatives to antibiotics, next-generation probiotics (NGPs) have emerged as a promising option. The genus Blautia presents a rich source of potential NGP strains. Here we successfully isolated Blautia hominis LYH1 strain from the intestines of healthy weaned piglets and characterized its biological traits. Its anti-inflammatory activity was then assessed using macrophages, while its protective effects against colitis and gut barrier damage were validated in a DSS-induced mouse colitis model.

Results: B. hominis LYH1 displayed typical characteristics of an obligate anaerobe, including non-hemolytic and non-motile features, and a genome enriched with carbohydrate-active enzyme genes. It produced metabolites with antibiotic-like compounds, demonstrating antimicrobial activity against Escherichia coli. In vitro, B. hominis LYH1 effectively inhibited pathogen replication in macrophages, reducing cellular infections and alleviating inflammatory damage. In vivo, oral administration of B. hominis LYH1 or its metabolites significantly mitigated DSS-induced colitis in mice by suppressing pro-inflammatory cytokines, inhibiting T-lymphocyte activation, and enhancing short-chain fatty acid production.

Conclusions: Our findings underscore B. hominis LYH1's potential as a NGP for maintaining gut health and combating intestinal inflammation. These findings offer valuable insights into the development of antibiotic alternatives and innovative strategies for preventing and treating enteritis in both agricultural and medical settings.

Keywords: Blautia hominis; Colitis; Inflammation; Macrophage.

Fig. 1

Morphological, hemolytic, and whole-genome of B. hominis LYH1. A Phylogenetic tree based on 16S rDNA. B Electron micrograph at 40,000 × magnification. C Growth pattern on blood agar plates. D Circular genome map. E KEGG clustering annotation (http://www.genome.jp)

Fig. 2

Metabolite profile of B. hominis LYH1 and antimicrobial activity of metabolites in co-cultures with pathogens. A–E Production of SCFAs (Acetic acid, Butyric acid, Valeric acid, Isovaleric acid, Total SCFA) in BHI medium. F and G Volcano plots of metabolite fold changes (|log₂FC| > 1) and significance (P > 0.05) between B and BHI media (n = 3). H and I PCA plots of cationic and anionic metabolite profiles. J KEGG pathway enrichment analysis (n = 3). K The preparation and co-culture process of B. hominis LYH1 metabolites. The tubes on the left and right serve as positive controls with pathogens, while the middle two tubes contain the metabolite treatment group. L–N Growth curves of E. coli, S. Typhimurium, and S. aureus with and without the addition of metabolites (n = 3). BHI Blank BHI medium, B B. hominis LYH1. Error bars represent standard deviation, and asterisks denote statistical significance: ^*P < 0.05, ^**P < 0.01, and ^***P < 0.001

Fig. 3

Effects of B. hominis LYH1 and metabolites on RAW264.7 macrophage viability, S. Typhimurium intracellular replication, and immune-related mRNA expression. A and B RAW264.7 macrophage viability with live bacteria or metabolites (n = 6). CON Control, 0.025×/0.05×/0.1× Respectively refer to live bacteria and their metabolites of B. hominis LYH1 at different concentrations. ^*P < 0.05, ^**P < 0.01. C and D Effects of live bacteria and metabolites at different concentrations on S. Typhimurium intracellular replication (n = 6). CON Control, 0.025×/0.05×/0.1× Respectively refer to live bacteria and their metabolites of B. hominis LYH1 at different concentrations. ^***P < 0.001. E and F Flow cytometry histograms depicting the effects of B. hominis LYH1 live bacteria and metabolites on S. Typhimurium-infected cells, measured by ΔMFI (n = 6). CON Control, 0.025×/0.05×/0.1× Respectively refer to live bacteria and their metabolites of B. hominis LYH1 at different concentrations. G–L mRNA expression levels of Il6, Il10, Tnf, Il17a, Nos2, and Tgfb1 in RAW264.7 macrophages treated with different concentrations of metabolites treatments (n = 6). CON Control, ST S. Typhimurium infection, 0.025×/0.05×/0.1× Cells infected with S. Typhimurium were treated with metabolites of B. hominis LYH1 at different concentrations. Identical lowercase letters indicate no significant difference between groups, while differing letters denote significant differences

Fig. 4

Effects of B. hominis LYH1 and metabolites on growth performance, colon length, spleen index, histopathological scores, and physical and chemical barriers in DSS-induced colitis mice. A Experimental design. B Body weight changes during DSS treatment (n = 10). C Disease Activity Index (DAI) scores (n = 10). D Representative images of cecum and colon from each group. E Colon length measurements across groups (n = 10). F Representative images of colonic morphology from each group, with hematoxylin and eosin (HE) staining; the upper row shows images at 4 × magnification, and the lower row shows images at 10 × magnification. G Representative images of colonic mucus layer thickness across groups, with Alcian Blue (AB) and nuclear fast red staining at 20 × magnification. H Histopathological scores of colonic tissues across groups (n = 10). I Measurements of colonic mucus layer thickness across groups (n = 10). J Spleen index (spleen weight/body weight) across groups (n = 10). K–M Relative mRNA expression levels of tight junction proteins Ocln, Cldn1 and Tjp1 in colonic tissues across groups (n = 10). N Western blot (WB) bands of tight junction proteins in colonic tissues across groups (n = 2). O–Q Protein expression levels of tight junction proteins OCLN, CLDN1 and TJP1 in colonic tissues across groups (n = 3, from two separate WB experiments). CON Control, DSS Administered 3% DSS, B Treated with 3% DSS and B. hominis LYH1 at a concentration of 10⁹ CFU/mL, B-M Exposed to 3% DSS alongside metabolites derived from B. hominis LYH1 at an equivalent concentration. ^*P < 0.05, ^**P < 0.01, and ^***P < 0.001. Identical lowercase letters indicate no significant difference between groups, while differing letters denote significant differences

Fig. 5

Effects of B. hominis LYH1 and metabolites on T lymphocytes and immune gene expression in DSS-colitis mice. A Representative flow cytometry plots from each group. B Proportions of T lymphocyte subsets, including CD3⁺, CD3⁺CD4⁺, CD3⁺CD8⁺, and the CD3⁺CD4⁺/CD3⁺CD8.⁺ ratio (n = 10). C and D Relative expression levels of pro-inflammatory and anti-inflammatory cytokine genes in the colons of mice across groups (n = 10). CON Control, DSS Administered 3% DSS, B Treated with 3% DSS and B. hominis LYH1 at a concentration of 10⁹ CFU/mL, B-M Exposed to 3% DSS alongside metabolites derived from B. hominis LYH1 at an equivalent concentration.^*P < 0.05, ^**P < 0.01, and ^***P < 0.001

Fig. 6

Effects of B. hominis LYH1 and metabolites on SCFA levels and gut microbiota in DSS-Colitis mice. A SCFA concentrations in colonic contents (n = 6). AA Acetic acid, PA Propionic acid, BA Butyric acid, IVA Isovaleric acid, VA Valeric acid, Total SCFA Total short-chain fatty acids. B α-Diversity indices (Chao1, Faith_PD, Observed_features, Shannon_entropy, Simpson). n = 6. C PLS-DA scores plot illustrating β-diversity of gut microbiota at the genus level (n = 6). D Relative abundance of gut microbiota at the phylum level for each treatment group. E Relative abundance of the top 20 gut microbial taxa at the genus level. F LEfSe analysis of gut microbiota communities at the genus level, highlighting significant taxa with LDA > 4. G Heatmap representing Spearman correlations between differential genera and SCFA concentrations. CON Control, DSS Administered 3% DSS, B Treated with 3% DSS and B. hominis LYH1 at a concentration of 10⁹ CFU/mL, B-M Exposed to 3% DSS alongside metabolites derived from B. hominis LYH1 at an equivalent concentration. Significant correlations are indicated by asterisks: *P < 0.05, **P < 0.01, and ***P < 0.001