Genomic characteristics and phylogenetic relationships of B. velezensis HBXN2020.

(A) The whole-genome map of B. velezensis HBXN2020 with its genomic features. The map consists of 6 circles. From the inner circle to the outer circle: (1) GC-skew, (2) GC content, (3) reverse protein-coding genes, different colors represents different COG functional classifications, (4) genes transcribed in reverse direction, (5) genes transcribed in forward direction, (6) forward protein-coding genes, different colors represents different COG functional classifications. (B) The whole genome phylogenetic tree was constructed based on genome-wide data from 14 bacillus strains. B. velezensis HBXN2020 are indicated in bold. (C) The heatmap based on the ANIb value of strain HBXN2020 and other Bacillus species. The B. velezensis HBXN2020 was labeled in red letters.

Growth curve of B. velezensis HBXN2020 and its in vitro resistance against environmental assaults.

(A) Growth curves of B. velezensis HBXN2020 cultured in LB medium at 37°C, detection of OD600 values at 1 h intervals in microplate reader. (B) Survival rate of endospore and vegetative cells of B. velezensis HBXN2020 after 30 min at different temperatures (37°C, 45°C, 55°C, 65°C, 75°C, 85°C or 95°C). Equal amounts of endospore and vegetative cells of HBXN2020 were exposed to the following: (C) acid solution (pH 2 to 7), (D) 0.3% bile salts, (E) SGF (pH 1.2) supplemented with pepsin, and (F) SIF (pH 6.8) containing trypsin at 37°C. At predetermined time points, 100 μL was taken from each sample, and tenfold serially diluted with sterile PBS (pH 7.2), and then spread on LB agar plates and cultured at 37°C for 12 h before bacterial counting. Each group was repeated three times (n = 3).

Antibiotic susceptibility of B. velezensis HBXN2020 and bacteriostasis assay in vitro.

(A) The diameter of the antibacterial zone indicates the extent of sensitivity to antibiotics. (B) The diameter of the antibacterial zone indicates the extent of inhibition against pathogenic bacteria. The diameter of the antibacterial zone was measured with vernier caliper. Each group was repeated three times (n = 3). R, resistant; I, moderately sensitive; S, sensitive.

In vivo safety evaluation of B. velezensis HBXN2020 in a mouse model.

(A) Body weights changes of mice during gavage with B. velezensis HBXN2020 spores. Mice were treated with sterile PBS (Control group) or low-dose (L-HBXN2020 group), medium dose (M-HBXN2020 group), and high-dose (H-HBXN2020 group) of B. velezensis HBXN2020 spores. Weighing and gavage were performed once every two days during the experimental period (15 days). Data were shown as mean values ± SEM (n = 5). (B) The relative gene expression levels of inflammatory cytokines in the colon of mice measured by RT-qPCR. Data were shown as mean values ± SEM (n = 5). (C) The relative gene expression levels of barrier protein ZO-1, occludin, claudin and Muc2 in the colon of mice measured by RT-qPCR. Data were shown as mean values ± SEM (n = 5). (D) Major blood routine parameters and (E) serum biochemical parameters of mice in the control group and H-HBXN2020 group. Data were shown as mean values ± SEM (n = 3). (F) H&E stained colon sections in the different groups. Scale bar: 200 μm.

Oral B. velezensis HBXN2020 spores alleviated S. Typhimurium-induced experimental mouse colitis.

(A) In vitro Bacterial competition between STm and B. velezensis HBXN2020. STm were co-incubated with B. velezensis HBXN2020 at various ratios at 37°C with shaking. The growth of STm was reflected by bacterial counting per hour. (B) Experimental design for treatment in this study. Orally administrated with either PBS, or B. velezensis HBXN2020 spores by gavage at days 1, 3, and 5 after STm infection (5× 107 CFU/mouse), respectively. All mice were euthanized at day 7 after STm infection. (C) Bacterial count of STm in mouse feces. Fecal samples were collected per day after STm infection and resuspended in sterile PBS (0.1 g of fecal resuspended in 1 mL of sterile PBS). One hundred microliters of each sample performed a serial of 10-fold dilutions and spread on selective agar plates (50 µg mL-1 kanamycin) and incubated at 37°C for 12 h before bacterial counting. The bacterial loads of STm in (D) cecum, and (E) colon. The cecum and colon were harvested and then homogenized. One hundred microliters of each sample performed a serial of 10-fold dilutions and spread on selective agar plates (50 µg mL-1 kanamycin) and incubated at 37°C for 12 h before bacterial counting. Statistical signifcance was evaluated using Student’s t-test (*, P < 0.05, **, P < 0.01, and ***, P < 0.001). (F) Daily body weight changes and (G) daily disease activity index (DAI) scores of mice with different treatment groups. Data were shown as mean values ± SEM (n = 8). Statistical signifcance was evaluated using one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test. *, P < 0.05, **, P < 0.01, and ***, P < 0.001 relative to Control group; #, P < 0.05, ##, P < 0.01, ###, P < 0.001 relative to STm + HBXN2020 group. (H) Colonic tissue images. (I) The length of the colon from per group (n = 8). Statistical signifcance was evaluated using one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test (*, P < 0.05, **, P < 0.01, and ***, P < 0.001).

Oral B. velezensis HBXN2020 spores attenuated colonic damage and inflammatory reaction.

(A) H&E stained colon tissue sections. Scale bar: 200 μm. The relative gene expression levels of TNF-α (B), IL-1β (C), IL-6 (D), and IL-10 (E) were detected by RT-qPCR. Data were shown as mean values ± SEM (n = 6). The relative gene expression levels of ZO-1 (F), ocludin (G), claudin (H), and Muc2 (I) in colon tissue were detected by RT-qPCR. Data were shown as mean values ± SEM (n = 6). Statistical signifcance was evaluated using one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test (*, P < 0.05, **, P < 0.01, and ***, P < 0.001).

Oral B. velezensis HBXN2020 spores regulated the composition of intestinal microbiota.

(A to D) The α-diversity of the gut microbiota, determined by the (A) Sobs, (B) Chao1, (C) Shannon, and (D) Simpson diversity index. Data were shown as mean values ± SEM (n = 5). (E) The PCA plot showed the β-diversity of the gut microbiota based on Bray-Curtis distance at the OTU level. (F and G) The relative abundance of colonic microbiota at the phylum (F) and genus (G) levels. (H-K) Relative abundance of selected taxa (H) norank_f_Muribaculaceae, (I) Lactobacillus, (J) Bacteroides, and (K) Escherichia-Shigella. Data were shown as mean values ± SEM (n = 5). (L) Analysis of differences in the microbial communities by LEfSe (linear discriminant analysis (LDA) score > 3.5) among different groups. Signifcance was evaluated by the Kruskal-Wallis test or ANOVA with Tukey’s multiple comparisons test (*, P < 0.05, **, P < 0.01, and ***, P < 0.001).

Prophylactic B. velezensis HBXN2020 spores attenuated the symptoms of S. Typhimurium-induced mouse colitis.

(A) Experimental design for treatment in this study. At days 1, 3, 5, and 7, each mouse in the HBXN2020 + STm group and PBS + STm group were received 200 μL (1×108 CFU/mouse) of B. velezensis HBXN2020 spores or sterile PBS by gavage. Then, mice in PBS + STm group and HBXN2020 + STm group were orally inoculated with 200 μL (5×107 CFU/mouse) of STm on day 7. On day 12, all mice were euthanized. (B) Daily body weight changes and (C) daily disease activity index (DAI) scores of mice with different groups following STm treatment. Data were shown as mean values ± SEM (n = 8). Statistical signifcance was evaluated using one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test. *, P < 0.05, **, P < 0.01, and ***, P < 0.001 relative to Control group; #, P < 0.05, ##, P < 0.01, ###, P < 0.001 relative to HBXN2020 + STm group. (D) Colonic tissue images. (E) The length of the colon from per group (n = 8). (F) Bacterial count of STm in mouse feces. Fecal samples were collected every day after STm infection and resuspended in sterile PBS (0.1 g of fecal resuspended in 1 mL of sterile PBS). One hundred microliters of each sample performed a serial of 10-fold dilutions and spread on selective agar plates (50 µg mL-1 kanamycin) and incubated at 37°C for 12 h before bacterial counting. The bacterial loads of STm in (G)cecum and (H)colon. The cecum and colon were harvested and then homogenized. One hundred microliters of each sample performed a serial of 10-fold dilutions and spread on selective agar plates (50 µg mL-1 kanamycin) and incubated at 37°C for 12 h before bacterial counting. Statistical signifcance was evaluated using Student’s t-test (*, P < 0.05, **, P < 0.01, and ***, P < 0.001).

Prophylactic B. velezensis HBXN2020 spores attenuated colonic damage and inflammatory reaction.

(A) H&E stained colon tissue sections. Scale bar: 200 μm. The relative gene expression levels of TNF-α (B), IL-1β (C), IL-6 (D), and IL-10 (E) were detected by RT-qPCR. Data were shown as mean values ± SEM (n = 6). The relative gene expression levels of ZO-1 (F), ocludin (G), claudin (H), and Muc2 (I) in colon tissue were detected by RT-qPCR. Data were shown as mean values ± SEM (n = 6). Statistical signifcance was evaluated using one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test (*, P < 0.05, **, P < 0.01, and ***, P < 0.001).

Prophylactic B. velezensis HBXN2020 spores regulated the composition of gut microbiota.

(A to D) Alpha diversity of the intestinal microbiota. Data were shown as mean values ± SEM (n = 5). (E) The PCA plot showed the β-diversity among different microbial community groups based on Bray-Curtis distance at the OTU level. (F and G) The relative abundance of colonic microbiota at the phylum (F) and genus (G) levels. (H-M) Relative abundance of selected taxa (H) Lactobacillus, (I) Akkermansia, (J) Alistipes, (K) Bacteroides, (L) Escherichia-Shigella, and (M) Enterococcus. Data were shown as mean values ± SEM (n = 5). (N) Analysis of differences in the microbial taxa by LEfSe (LDA score > 3.5) in different groups. Signifcance was evaluated by the Kruskal-Wallis test or ANOVA with Tukey’s multiple comparisons test (*, P < 0.05, **, P < 0.01, and ***, P < 0.001).