Additional file 1 of Vitamin B12 produced by Cetobacterium somerae improves host resistance against pathogen infection through strengthening the interactions within gut microbiota

Additional file 1: Figure S1. Identification, hemolytic assay and antibiotics susceptibility of Bacillus velezensis strain 1704-Y (BV1704-Y). a The colony characteristics of BV1704-Y in LB agar plate. b Gram stain status and morphological features of BV1704-Y. c Phylogenetic tree constructed by the neighbor-joining method based on the gyrB gene sequences. d Hemolytic assay for BV1704-Y. e Antibiotics susceptibility testing of BV1704-Y by the paper disc diffusion assay (1-Ofloxacin; 2-Ampicillin; 3-Ceftazidime; 4-Erythromycin; 5-Chloramphenicol; 6-Polymixin B). The detailed information on antibiotics susceptibility of BV1704-Y is listed in Additional file 2: Table S2. Figure S2. Dietary supplemented with Bacillus velezensis induced the change of gut microbiota composition in zebrafish. Simpson index (a) and number of observed OTUs (b) comparison among the four different groups (Y, CK, TY and TCK). c Relative abundance of the top 6 phyla in the gut samples from the four different groups. (d) The relative abundances of Fusobacteria in the gut sample from Y and CK. (e) The relative abundances of Fusobacteria in the gut sample from TY and TCK. (f) Different bacterial taxa enriched in the TY and TCK zebrafish by LEfSe (LDA score [log 10] > 3.0). (g-r) Relative abundance of selected different taxa. Data are expressed as box plot. *P < 0.05, **P < 0.01 by Mann–Whitney U test with Bonferroni-adjusted P-values. (s) Relative abundance of Cetobacterium after antibiotics treated. The statistical difference was examined using Kruskal–Wallis H-test followed by Dunn’s multiple comparisons test with Bonferroni-adjusted P-values. Figure S3. Heat map of Pearson's correlation coefficients between the top 20 genera and infection status (Y:TY). Dark red indicates a stronger positive correlation, dark blue indicates a stronger negative correlation, and white indicates no correlation. Black asterisk (*) means FDR-corrected P-value < 0.05. Figure S4. Distribution of genes across KEGG functional categories in the genome of C. somerae CS2105-BJ. Figure S5. C. somerae CS2105-BJ produces B12 both in vitro and in vivo. a The B12 production of CS2105-BJ strain in different growth phases. b The content of B12 in the gut of fish sampled prior to bath infection. * means FDR-corrected P-value < 0.05. c The liner regression between the content of B12 and the abundance of Cetobacterium in the gut of fish (CK and Ceto). r and P were obtained by Pearson’s correlation analysis. Figure S6. a Vitamin B12 content in the intestine of zebrafish in different treatment groups. b survival rate of zebrafish in different groups after infection with Aeromonas hydrophila. Figure S7. The protective effect of B12 on zebrafish is concentration-dependent. a The content of B12 in the gut of fish sampled prior to bath infection. CK: The zebrafish were fed a basic diet; 50: The zebrafish were fed a basic diet supplemented with vitamin B12 (50 μg/kg diet per day); 100: The zebrafish were fed a basic diet supplemented with vitamin B12 (100 μg/kg diet per day); 200: The zebrafish were fed a basic diet supplemented with vitamin B12 (200 μg/kg diet per day). Significant differences (P < 0.05) between different groups are indicated with different lowercase letters above the bars. b Kaplan–Meier graph of the zebrafish survival in different groups after bath infection with A. hydrophila. * indicates significant difference (P < 0.05) between different groups. Figure S8. Vitamin B12 induces alterations in gut microbiota structure. Microbiota alpha diversity was measured by 16S rRNA gene sequence analysis of the gut content samples using Shannon index, Chao1 index, and Observed_otus. Error bars were median with interquartile ranges. * P-value < 0.05. Figure S9. Vitamin B12 induces alterations in gut microbiota structure and function. a Principal coordinate analysis (PCoA) of bray curtis distance was analyzed based on OTU level for microbiota beta diversity (ANOSIM R = 0.7893, P = 0.001). b Phylum-level taxonomic distributions of the microbial communities in gut of zebrafish fed with different diets. c Liner discriminant analysis effect size (LEfSe) was used to analyze the difference in microbial abundance between TB and TCK group, The LDA value threshold was set at 4.0. d Bacterial community phenotypes of the gut microbiome were predicted using BugBase. Statistical significance was identified by the Wilcoxon test with false discovery rate (FDR)-corrected pairwise P values. *, P < 0.05. e Functional alterations of the gut microbiome in zebrafish fed with control (TCK) and B12-supplementd diet (TB) after infection with A. hydrophila. Statistical significance was determined by using LEfSe, with a P value of < 0.05 (Wilcoxon test) and a linear discriminant analysis (LDA) score (log10) of > 2.2 being considered significant. Figure S10. The heatmap shows relative abundance changes for the bacterial genera among the 50 most abundant in any sample. The relative values in the heatmap (after normalization), represented by colors, show the distribution of bacterial species at the genus level among the samples. Red color represents higher abundance, and blue lower abundances. Figure S11. B12 affects the relative abundance of anaerobic microbiota in gut. Pearson linear correlation between the relative abundance of anaerobic microbiota and vitamin B12 quantification of zebrafish fed with control and B12-supplemented diet in gut. Figure S12. B12 affects the gut redox potential. a Intestinal redox potential levels. b Pearson linear correlation between Redox potential and vitamin B12 quantification of zebrafish fed with control and B12-supplemented diet in gut. Figure S13. B12 affects the ecological network of gut microbiome. Demonstration of constructed molecular ecological networks generated using the Molecular Ecological Network Analysis (MENA) pipeline based on OTU relative abundances of gut microbiota. Each link denotes a correlation between two nodes, and each node stands in for a single OTU. Smaller network modules (between 2 and 5 nodes) are depicted in gray, whereas larger network modules (above 5 nodes) are shown in various colors. Figure S14. Network analyses for TCK and TB. Large modules (> 5 nodes) are shown in circular layout. Positive and negative correlations are indicated by red and green connections, respectively. The module ID of each large module is indicated by M1 to M11. Figure S15. Vitamin B12 affect the relative mRNA expression of Zo-1, Occludin, and Claudin15. Relative mRNA expression of Zo-1, Occludin, and Claudin15 in different groups. Data were normalized for β-actin expression and expressed as fold change. Values represent means ± SD. Significant differences (P < 0.05) between different groups are indicated with different lowercase letters above the bars. CK: The zebrafish were fed a basic diet; B: The zebrafish were fed a basic diet supplemented with vitamin B12; ACK: The zebrafish treated with antibiotics for 7 days prior to administration of basic diet; AB: The zebrafish treated with antibiotics for 7 days prior to administration of B12; TCK: The zebrafish were fed a basic diet and then bath infected with A. hydrophila; TB: The zebrafish were fed a basic diet supplemented with vitamin B12 and then bath infected with A. hydrophila. TACK: The zebrafish treated with antibiotics for 7 days prior to administration of basic diet, and then fed a basic diet and then bath infected with A. hydrophila; TAB: The zebrafish treated with antibiotics for 7 days prior to administration of basic diet, and then fed a basic diet supplemented with vitamin B12 and then bath infected with A. hydrophila. Figure S16. B. velezensis/Cetobacterium/Vitamin B12 affect the relative mRNA expression of Zo-1, Occludin, and Claudin15. Relative mRNA expression of Zo-1, Occludin, and Claudin15 in different groups. Data were normalized for β-actin expression and expressed as fold change. Values represent means ± SD. Significant differences (P < 0.05) between different groups are indicated with different lowercase letters above the bars. CK: The zebrafish were fed a basic diet; Y: The zebrafish were fed a basic diet supplemented with B. velezensis; B12: The zebrafish were fed a basic diet supplemented with vitamin B12; Ceto: The zebrafish were fed a basic diet supplemented with Cetobacterium; TCK: The zebrafish were fed a basic diet and then bath infected with A. hydrophila; TY: The zebrafish were fed a basic diet supplemented with B. velezensis and then bath infected with A. hydrophila; TB12: The zebrafish were fed a basic diet supplemented with vitamin B12 and then bath infected with A. hydrophila; TCeto: The zebrafish were fed a basic diet supplemented with Cetobacterium and then bath infected with A. hydrophila.