The bacterial strains used in this study are listed in Supplementary Table S1, and plasmids and primers are listed in Supplementary Table S2. The streptococci strains were cultured in tryptic soy broth (TSB) (BD Biosciences, MD, United States) supplemented with 5% (v/v) fetal bovine serum (Solarbio, Beijing, China) or TSB plates containing 1.5% (w/v) agar and 5% (v/v) fetal bovine serum at 37°C. The other strains were cultured in Luria–Bertani (LB) broth or LB plates containing 1.5% (w/v) agar at 37°C. B. subtilis sporulation was cultured in Difco sporulation medium (DSM), as previously described (Mascarenhas et al., 2002).

To display phage lysin 0859 (Lys0859), which was previously discovered in our laboratory (Li et al., 2023), on the surface of B. subtilis 168 spores, we constructed a CotG-0859 fusion. The CotG gene was amplified with primers CotG-F/CotG-R from the B. subtilis 168 chromosomes. The Lys0859 gene from S. suis 0859 was amplified using primers 0859-F/0859-R by PCR. Then, the CotG and 0859 genes were constructed into a CotG-0859 fusion by overlap-extension PCR, digested with BamH I and EcoR I (Takara, China), and ligated into pDG364 vector to obtain the recombinant plasmids pDG364-CotG-0859. Meanwhile, pCold-CotG-0859 (CotG-F/CotG-R, 0859-F/0859-R1, BamH I/Hind III) and pCold-0859 (0859-F1/0859-R1, BamH I/Hind III) were constructed using the same methods. Recombinant plasmids were transformed into E. coli DH5α and the positive colonies were identified by colony-PCR.

The obtained pDG364-CotG-0859 recombinant plasmid was linearized by Kpn I (Takara, China) enzyme digestion, and transformed into the amylase E (AmyE) gene of the competent B. subtilis genome by the natural transformation method (Spizizen, 1958). Then, the clones generated after the integration of the target gene at the AmyE locus of B. subtilis were selected on LB agar plates supplemented with 5 μg/mL chloramphenicol. Used LB agar plates containing 1% starch to screen positive strains with amylase gene deletion. Extracted recombinant bacterial genomic DNA and used AmyE-F/AmyE-R, G8-F/G8-R, G8-F/AmyE-R, and AmyE-F/G8-R for PCR identification, respectively.

The sporulation of wild-type B. subtilis 168 (WT) and recombinant B. subtilis were induced at 37°C for 48 h using Difco Sporulation Medium (DSM). Spore purification was performed as previously described, with some modifications. The spores were collected by centrifugation 8,000 g for 10 min at 4°C, and washed with 0.5 M NaCl. Resuspend the spores in Tris–HCl (50 mmol/L, pH 7.2) containing a final concentration of 50 μg/mL of lysozyme and incubate at 37°C in a water bath for 1 h. Then, the spores were harvested by centrifugation and washed with 1M NaCl, 1M KCl, and distilled water (three times), respectively. After incubation at 65°C for 1 h, pure spores were harvested and resuspended in sterile PBS, and stored at −40°C.

The purified spores were incubated with SDS-DTT extraction buffer at 70°C for 30 min to extract spore coat proteins. To confirm that the surface display of Lys0859 on the spore coat, the extracted spore coat proteins was subjected to SDS-PAGE. Subsequently, one portion was stained with Coomassie Brilliant Blue, while the other portion was transferred to polyvinylidene fluoride (PVDF) membrane. The membrane was incubated with PBST containing 5% skim milk for 2 h. After three washes with PBST, incubates with Lys0859 antiserum (1:5,000 in PBST) for 2 h. Then, the membrane was incubated with HRP-conjugated Goat anti-Mouse IgG second antibody (1:5,000 in PBST) for 1 h at room temperature and visualized by ECL detection.

To detect Lys0859 on the surface of spores, 1 mL of purified spore solution was harvested after 48 h of B. subtilis induction and fixed on a glass slide, and blocked with 5% BSA for 2 h. Anti-Lys0859 antibody (1:400 in PBST) was added and incubated for 2 h at room temperature. Then, FITC labeled goat anti-mouse IgG antibody (1:400 in PBST) was added and the spores were observed with a fluorescence microscope (Olympus, Japan).

To investigate whether the insertion of exogenous proteins affects the activity of the strain, we determined the growth curves and sporulation rate of B. subtilis 168 and rBS^CotG-0859. B. subtilis 168 and rBS^CotG-0859 were inoculated into LB medium or DSM sporulation medium (final concentration 1 × 10⁴ CFU/mL) respectively, and incubated at 37°C with shaking (180 rpm). Samples were collected every 2 h to prepare 10-fold serial dilutions and spread onto LB agar plates and cultured at 37°C for 12 h before bacterial counting. For spore counting, the spore suspensions were treated at 85°C for 5 min, and then 10-fold serial dilutions were prepared to place on LB agar plates and cultured at 37°C for 12 h before counting. The sporulation rate was calculated with the following equation: Sporulation rate = number of spores/ number of vegetative cells.

The rBS^CotG-0859 spores (1 × 10⁷ CFU) were separately resuspended into 1 mL of sterile PBS with different pH values (1, 2, 3, 4, 5, 6, 7, or 8), or simulated gastric fluid (SGF, HCl, pH 1.2) containing 10 g/L of pepsin in 0.85% NaCl solution, or simulated intestinal fluid (SIF, NaOH, pH 6.8) containing 10 g/L of trypsin in 0.05 M KH₂PO₄ solution, and incubated at 37°C. At predetermined time points, the samples were centrifuged at 6,000 g for 5 min at 4°C, and bacterial cells were washed twice with sterile PBS and resuspended in 100 μL of PBS. Next, bacteriostatic activity of rBS^CotG-0859 spores (1 × 10⁷ CFU) against S. suis SC19 was tested by the agar-well diffusion assays. The plates were incubated at 37°C for 12 h, and the diameter of inhibition zone was measured with vernier caliper.

One milliliter of rBS^CotG-0859 spores (1 × 10⁷ CFU) were separately placed in water baths at different temperatures (37°C, 45°C, 55°C, 65°C, 75°C, 85°C, or 95°C) for 30 min. Bacterial cells were then collected by centrifugation (6,000 g, 5 min, 4°C), washed twice and resuspended in 100 μL sterile PBS. The bacteriostatic activity was measured as described above.

To assess the genetic stability of recombinant bacteria, the rBS^CotG-0859 were serially passaged on LB agar plates up to 10 times. A single bacterial colony was picked and inoculated into 1 mL of LB medium (contains 5 μg/mL chloramphenicol) and incubated overnight at 37°C with shaking (180 rpm). PCR identification of rBS^CotG-0859 clone was performed using 168-F/168-R and G8-F/G8-R primers, respectively. The rBS^CotG-0859 (the 1st to 10th generation colonies) were induced to sporulate in DSM for 48 h at 37°C and purified them. The antibacterial activity of rBS^CotG-0859 (the 1st to 10th generation) against S. suis SC19 by agar diffusion assay. All plates were cultured at 37°C for 12 h before observing the inhibition zone, and the diameter of the inhibition zone was measured using a vernier caliper. The presence of a clear zone indicated antagonistic activity.

The in vitro antagonistic activity of rBS^CotG-0859 spores was tested using the agar well-diffusion method against common pathogens. The bacterial lawns were prepared by mixing 10 mL of TSB medium containing 5% fetal bovine serum (FBS) with bacterial culture suspension (1 × 10⁶ CFU/mL) and then poured onto a sterile plate covered with LB agar and Oxford cups, wells of 8 mm diameter were made on TSB agar after removing the cups. The rBS^CotG-0859 spores and different dosages of Lys0859 in a total volume of 100 μL were then added separately into the well on the agar plate. After incubating at 37°C for 12 h, and measuring the diameter of inhibition zone. The diameter of the inhibition zone (mm) was the average of three independent experiments and presented in the form of a bar graph. The five known doses of Lys0859 and their corresponding inhibition zone sizes were displayed on the x-y scatter plot, respectively. Thereafter, the best-fit linear regression equation (LRE) was plotted based on the linear trend line. After inhibition zone diameter (rBS^CotG-0859) were fitted in the LRE, the equivalent dose of lysis enzyme activity of the recombinant bacteria was ultimately obtained.

A model of S. suis SC19 infection mouse was established using 4-week-old specific pathogen-free (SPF) female ICR mice purchased from the Experimental Animal Center of Huazhong Agricultural University, Wuhan, China. Mice were randomly divided into 4 groups (n = 6): (i) control; (ii) SC19 + PBS group; (iii) SC19 + BS168; (iiii) SC19 + rBS^CotG-0859. On the first experimental day, all mice were injected intraperitoneally with 100 μL of SC19 (6 × 10⁷ CFU/mouse) in the SC19 + PBS group, SC19 + BS168 group, and SC19 + rBS^CotG-0859 group. On days 1, 2, 3 and 4 following SC19 infection, mice in the SC19 + rBS^CotG-0859 group and SC19 + BS168 group separately received 200 μL (2 × 10⁷ CFU/mouse) of rBS^CotG-0859 spores and BS168 spores via gavage administration. In contrast, the mice in the control group and the SC19 + PBS group were administered the same volume of sterile PBS. Fresh fecal samples were collected daily following SC19 infection and directly resuspended in sterile PBS. The number of rBS^CotG-0859 in mice feces from the SC19 + rBS^CotG-0859 group was then determined by spreading a series of 10-fold dilutions on LB agar plates containing 10 μg/mL chloramphenicol. The general health of all mice was monitored on a regular daily throughout the experiment. On the 7th day after SC19 infection, all mice were sacrificed by cervical vertebra dislocation, the blood and the major organs (heart, liver, spleen, lung, kidney, and brain) were collected and fixed with 4% paraformaldehyde. Then, the remaining organ tissues were weighed and homogenized. The number of SC19 in the heart, liver, spleen, lungs, kidneys, brain, and blood were determined by spreading a series of 10-fold dilutions on TSA plates.

To further study the prophylactic efficacy of rBS^CotG-0859 spores against SC19 infection, another independent experiment was conducted using four-week-old female ICR mice (SPF). Twenty-four mice were randomly divided into four groups (n = 6): control group, PBS + SC19 group, BS168 + SC19 group and rBS^CotG-0859 + SC19 group. On days 1 to 7, each mouse in the rBS^CotG-0859 + SC19 group received 200 μL (2 × 10⁷ CFU/mouse) of rBS^CotG-0859 spores via gavage once a day. At the same time, mice in the BS168 + SC19 group received an equal amount of BS168 spores, while intragastric gavages with 200 μL of sterile PBS to mice of the control group and PBS + SC19 group. On day 7, all mice in the PBS + SC19 group, BS168 + SC19 group, and rBS^CotG-0859 + SC19 group were injected intraperitoneally with 200 μL of SC19 (6 × 10⁷ CFU/mouse). Fresh fecal samples were collected daily following SC19 infection and directly resuspended in sterile PBS. The number of rBS^CotG-0859 in feces of mice from the rBS^CotG-0859 + SC19 group was determined referring to the method described in the rBS^CotG-0859 therapeutic trial. Throughout the study, the health of the mice was monitored on a regular daily. On day 12, all mice were sacrificed by cervical vertebra dislocation, the blood and the major organs (heart, liver, spleen, lung, kidney, and brain) were collected and fixed with 4% paraformaldehyde. Then, the remaining organ tissues were weighed and homogenized, and the number of SC19 in the heart, liver, spleen, lungs, kidneys, brain, and blood was measured using TSA plates.

All animal experiments were performed with the approval of the Scientific Ethic Committee of Huazhong Agricultural University (no. HZAUMO-2024-0075).

Statistical analysis was performed using GraphPad Prism 8.3.0 (GraphPad Software, San Diego, CA, United States). Data were expressed as mean ± standard deviation (SD), and analysis comparisons were carried out using one-way analysis of variance (ANOVA) followed by Tukey’s multiple-comparison test (*p < 0.05, **p < 0.01, and ***p < 0.001).

To validate whether Lys0859 displayed on the spore surface enhances antibacterial activity or tolerance, we constructed a recombinant B. subtilis strain (rBS^CotG-0859) expressing Lys0859 on its spore surface. Briefly, the exogenous fusion gene CotG-0859 was inserted into the amylase gene of B. subtilis through homologous recombination, resulting in the inactivation of the amylase gene (Figure 1a). Therefore, recombinant bacteria (rBS^CotG-0859) could not hydrolyze starch compared to the wild-type (Figure 1b). To further verify whether CotG-0859 was successfully inserted into the AmyE gene, PCR were performed using 4 sets primers. The results of agarose-gel electrophoresis showed that the bands amplified from the genome of rBS^CotG-0859 were 4,448 bp, 1,544 bp, 3,831 bp, and 2,161 bp from left to right, which was consistent with the expected size (Figure 1c). We used Coomassie blue staining to identify the capsid proteins extracted from the rBS^CotG-0859 spores. A specific band with molecular weight of 51 kDa appeared in the extracts of rBS^CotG-0859 spores, but extract from wild strains (B. subtilis 168) did not (Figure 1d). In addition, western blot results showed that a positive hybridization band with 51 kDa was detected in the capsid proteins extracted from the rBS^CotG-0859 spores. Conversely, it was not detected in the extracts of wild strains (B. subtilis 168) (Figure 1e). The surface expression of the fusion protein was also confirmed by immunofluorescence (IF), with strong fluorescence was observed on rBS^CotG-0859 spores compared to wild-type spores (Figure 1f).

The growth curve and sporulation rate of B. subtilis 168 and rBS^CotG-0859 were determined through a standard plate-counting method. As shown in Figure 2a, the growth curve of the rBS^CotG-0859 was always consistent with the B. subtilis 168, indicating that the insertion of exogenous DNA did not affect the growth of the recombinant strains. In addition, the high-level expression of heterologous proteins did not have a significant effect on the sporulation rate of rBS^CotG-0859 compared with the B. subtilis 168 (Figure 2b).

Moreover, we evaluated the effects of temperature, pH, SGF, and SIF on the antibacterial activity of rBS^CotG-0859 spores. As shown in Figure 2c, the antibacterial activity of rBS^CotG-0859 spores against S. suis SC19 showed a gentle downward trend with increasing temperature. Meanwhile, the antibacterial activity of rBS^CotG-0859 spores showed a declining tendency toward when the pH decreased (Figure 2d), and still maintained excellent antibacterial activity at pH 1.0. Similar experimental results were observed in both simulated gastric fluid (pepsin 1 mg/mL, pH 1.2) (Figure 2e) and simulated intestinal fluid (trypsin 1 mg/mL, pH 6.8) (Figure 2f). Furthermore, the rBS^CotG-0859 were serially passaged on LB agar plates up to 10 times (Supplementary Figure S1), and identified by PCR analysis. Nucleic acid electrophoresis showed that the bands amplified from the 1st to 10th generation rBS^CotG-0859 genome were 1,544 bp and 4,854 bp (Figure 2g), which was consistent with the expected size. As shown in Figures 2h,i, the 1st to 10th generation rBS^CotG-0859 spores exhibited good antibacterial activity against S. suis SC19.

Following confirmation that the Lys0859 enzyme successful displayed on the surface of rBS^CotG-0859 spores, we validated whether the rBS^CotG-0859 spores exhibited bactericidal activity against S. suis SC19 through an agar-well diffusion assay. The purified rBS^CotG-0859 spores were added into an 8-mm diameter well on agar plate confluent with S. suis SC19, and incubated at 37°C for 12 h. The well containing Lys0859 enzyme displayed an antibacterial zone, served as a positive control, while the spore of B. subtilis 168 has no antibacterial activity and used as a negative control (Figure 3a). The inhibition zone diameters (11.56, 11.92, 12.17, and 12.41 mm, respectively) of Lys0859 enzyme with different dosages (from low to high were 20, 30, 40, and 50 μg) were measured on a double-layer agar plate containing S. suis SC19 (Figure 3c). To better understand the relationship between dose and inhibition zone, three known dosages of Lys0859 enzyme and corresponding inhibition zones were transformed into a linear regression equation, which is y = 0.0028x + 11.035, R² = 0.9896 (Figure 3b). After incorporating the inhibition zone diameters of rBS^CotG-0859 spores (1 × 10⁶ CFU) into the regression equation, the antibacterial potency against S. suis SC19 were equivalent to 39.11 μg Lys0859 enzyme, respectively.

Based on the excellent antibacterial effect of Lys0859 displayed by rBS^CotG-0859 against S. suis SC19, we next examined whether rBS^CotG-0859 spores could kill clinical isolates (pathogenic bacteria) of S. suis. Here we still use Lys0859 as a positive control for the agar well diffusion assay, and B. subtilis 168 spores (1 × 10⁷ CFU) as a negative control. The results of the study demonstrated that Lys0859 exhibited a dose-dependent antibacterial effect against streptocci. Four dosages of Lys0859 with concentrations of 20, 30, 40, and 50 μg showed an average inhibition zone of 11.2, 12.02, 12.41, 12.66 mm (Supplementary Figure S2a), 14.15, 14.77, 14.92, 15.25 mm (Supplementary Figure S2b), 12.95, 13.3, 13.73, 13.96 mm (Supplementary Figure S2c), 12.03, 12.31, 12.74, 12.92 mm (Supplementary Figure S2d), 11.22, 11.53, 11.86, 12.17 mm (Supplementary Figure S2e), 8.98, 9.25, 9.82, 9.93 mm (Supplementary Figure S2f), and 12.65, 12.98, 13.34, 13.57 mm (Supplementary Figure S2g), respectively, on the agar plate containing S. suis SS3, S. suis 18SS35, S. suis SS4, S. suis 18SS8, S. suis SS19, S. suis 1SS3 or S. suis 18SS75. The rBS^CotG-0859 spores with concentrations of 1 × 10⁶ CFU exhibited an average inhibition zone of 12.54, 14.34, 14.11 and 12.25 mm against S. suis SS3 (Supplementary Figure S2a), S. suis 18SS35 (Supplementary Figure S2b), S. suis SS4 (Supplementary Figure S2c) and S. suis 18SS8 (Supplementary Figure S2d), respectively. Additionally, the rBS^CotG-0859 spores at a concentration of 1 × 10⁵ CFU displayed mean inhibition zone of 11.78 mm, 10.86 mm, and 13.58 mm against S. suis SS19 (Supplementary Figure S2e), S. suis 1SS3 (Supplementary Figure S2f) and S. suis 18SS75 (Supplementary Figure S2g), respectively. Based on the linear regression analysis (Table 1), the bactericidal activity of 1 × 10⁶ CFU rBS^CotG-0859 spores against S. suis SS3, S. suis 18SS35, S. suis SS4 and S. suis 18SS8 were equivalent to 44.52 μg, 22.14 μg, 52.45 μg and 26.93 μg of Lys0859, respectively. The bactericidal activity of 1 × 10⁵ CFU rBS^CotG-0859 spores against S. suis SS19, S. suis 1SS3 and S. suis 18SS75 were equivalent to 37.44 μg, 75.35 μg, 49.58 μg of Lys0859, respectively.

For S. suis SS15, S. suis SS23, S. suis SS30, S. suis 18SS23, S. suis 18SS29, S. suis 18SS91 and S. suis 19SS9, four different dosages of Lys0859 (20, 30, 40, and 50 μg) exhibited an average inhibition zone of 13.86, 14.22, 14.51, 14.94 mm (Supplementary Figure S3a), 14.75, 15.11, 15.34, 15.49 mm (Supplementary Figure S3b), 13.67, 14.17, 14.35, 15.21 mm (Supplementary Figure S3c), 12.89, 13.16, 13.46, 13.89 mm (Supplementary Figure S3d), 13.87, 14.31, 14.66, 14.87 mm (Supplementary Figure S3e), 12.85, 13.22, 13.76, 14.32 mm (Supplementary Figure S3f), and 9.72, 10.38, 10.97, 11.21 mm (Supplementary Figure S3g), respectively. The rBS^CotG-0859 spores with concentrations of 1 × 10⁷ CFU showed an average inhibition zone of 13.57, 14.39, 12.85, 13.67, 14.43, 13.19 and 11.12 mm against S. suis SS15 (Supplementary Figure S3a), S. suis SS23 (Supplementary Figure S3b), S. suis SS30 (Supplementary Figure S3c), S. suis 18SS23 (Supplementary Figure S3d), S. suis 18SS29 (Supplementary Figure S3e), S. suis 18SS91 (Supplementary Figure S3f) and S. suis 19SS9 (Supplementary Figure S3g), respectively. Calculated based on the linear regression equation shown in Table 1, the bactericidal activity of 1 × 10⁷ CFU rBS^CotG-0859 spores against S. suis SS15, S. suis SS23, S. suis SS30, S. suis 18SS23, S. suis 18SS29, S. suis 18SS91 and S. suis 19SS9 were equivalent to 12.09 μg, 30 μg, 3.75 μg, 44.70 μg, 34.56 μg, 27.7 μg and 45.51 μg of Lys0859, respectively.

Next, we performed another antimicrobial test on Streptococcus agalactiae ATCC13813, Streptococcus agalactiae X2 and Streptococcus dysgalactiae SD002. Analogous to the above experiments, different dosages of Lys0859 (20, 30, 40, and 50 μg) exhibited an average inhibition zone of 9.95, 10.42, 10.83, 11.19 mm, 8.81, 9.46, 9.91, 10.21 mm, and 9.78, 10.42, 10.65, 10.87 mm on the agar plate containing S. agalactiae ATCC13813 (Supplementary Figure S4a), S. agalactiae X2 (Supplementary Figure S4b) or S. dysgalactiae SD002 (Supplementary Figure S4c), respectively. The average inhibition zones of rBS^CotG-0859 spores (1 × 10⁶ CFU) against S. agalactiae ATCC13813, S. agalactiae X2 and S. dysgalactiae SD002 were 10.21, 11.68, and 10.84 mm, respectively. The bactericidal activity of 1 × 10⁶ CFU rBS^CotG-0859 spores against S. agalactiae ATCC13813, S. agalactiae X2 and S. dysgalactiae SD002 was equivalent to 25.85 μg, 57.65 μg and 46.71 μg of Lys0859 through linear regression analysis.

We also validated the bactericidal activity of rBS^CotG-0859 spores against S. aureus ATCC43300 and found that Lys0859 with different dosages of 20, 30, 40, and 50 μg exhibited an average inhibition zone of 10.68, 11.38, 11.55, 11.95 mm (Supplementary Figure S4d), respectively. The rBS^CotG-0859 (1 × 10⁷ CFU) spores displayed an average inhibition zone of 10.79 mm on the agar plate containing S. aureus ATCC43300. Based on the results of a simple linear regression analysis, it was determined that the bactericidal activity of 1 × 10⁷ CFU rBS^CotG-0859 spores against S. aureus ATCC43300 was found to be equivalent to 19.83 μg of Lys0859 (Supplementary Figure S4d).

To evaluate the antibacterial efficacy of rBS^CotG-0859 in vivo, we challenged mice with the S. suis SC19 and treated with rBS^CotG-0859 spores by oral gavage (Figure 4a). As shown in Figure 4b, at least 10⁶ CFU/g of rBS^CotG-0859 was detected in feces of mice at day 1–4 after post-infection. Subsequently, the amount of rBS^CotG-0859 in mouse feces showed a progressively decreasing trend from day 5 to day 7. The treatment of rBS^CotG-0859 spores reduced the SC19 in the heart, liver, spleen, lungs, kidneys, brain, and blood of mice by 1.14, 1.53, 1.81, 1.96, 0.97, 0.47, and 0.52 logs, respectively (Figure 4c), compared to PBS-treat group. Meanwhile, the treatment of rBS^CotG-0859 spores decreased the SC19 in the heart, liver, spleen, lungs, kidneys, brain, and blood of mice by 0.93, 1.50, 1.43, 1.53, 1.07, 0.69, and 0.39 logs, respectively (Figure 4c), compared with the BS168 treatment group. Histology revealed that the rBS^CotG-0859 spores significantly relieved brain and lung inflammation and pathological damages such as inflammatory cell infiltrates, alveolar thickening, alveoli interstitial congestion, and edema in the brain and lung of infected mice (Figure 4d).

Based on the enhanced efficacy of rBS^CotG-0859 spores in the treatment of SC19 infection, the potential of disease prevention was further explored through pre-treatment (Figure 5a). As shown in Figure 5b, the number of rBS^CotG-0859 in mouse feces showed a gradually decreasing trend from day 8 to day 12 of the experiment, which is similar to the results of the therapeutic experiment. The prophylactic treatment of rBS^CotG-0859 spores reduced the SC19 in the heart, liver, spleen, lungs, kidneys, brain, and blood of mice by 1.42, 1.48, 0.77, 1.09, 1.21, 0.87, and 1.05 logs respectively, compared to the group pretreated with PBS (Figure 5c). Similarly, the prophylactic treatment of rBS^CotG-0859 spores declined the SC19 in the heart, liver, spleen, lungs, kidneys, brain, and blood of mice by 0.92, 1.25, 1.05, 1.21, 1.16, 0.38, and 0.75 logs respectively, compared with the BS168 pretreated group (Figure 5c). The results of histological analysis further that oral administration of rBS^CotG-0859 spores significantly reduced the severity of brain and lung injury (Figure 5d), consistent with the overall results of the therapeutic trial.