In vitro antibacterial efficacy of a novel chicken-derived Bacillus subtilis GX15 strain and its protective mechanisms in mice challenged by Salmonella enterica serovar typhymurium

Abstract

Background: The escalating issue of bacterial resistance, coupled with stringent restrictions on antibiotic use in many countries, has prompted the search for alternative strategies to combat bacterial infections. Probiotics, such as Bacillus subtilis (B. subtilis), have been widely recognized for their ability to inhibit pathogenic bacteria proliferation and protect hosts from infection-related damage.

Results: In this study, a novel strain of B. subtilis, designated GX15, was isolated from the intestine of a healthy chicken. We systematically evaluated its in vitro antimicrobial activity and protective effects against Salmonella Typhimurium SM022 (S. Typhimurium SM022) infection in mice. GX15 exhibited broad-spectrum antibacterial activity, effectively inhibited the growth of both Gram-positive and Gram-negative bacteria, including S. Typhimurium SM022, Escherichia coli, and Staphylococcus aureus. In a murine model, S. Typhimurium SM022 infection induced clinical symptoms such as diarrhea, anorexia, and fur ruffling in C57BL/6 mice. Oral administration of GX15 at 10⁸ CFU/mL significantly attenuated these effects, reducing the elevation of immune organ indices, limiting bacterial translocation to peripheral tissues, and alleviating histopathological damage to the liver and intestinal tissues. Notably, GX15 did not alter antioxidant indices or immunoglobulin levels. However, it markedly modulated the expression of cytokines including GM-CSF, IL-6, TLR2, IL-10, and TGF-β in the small intestine, suggesting an immunoregulatory mechanism of action.

Conclusion: B. subtilis GX15 exhibits potent probiotic activity and represents a promising prophylactic candidate against S. Typhimurium infections. These findings provide valuable insights into the potential use of GX15 as an alternative strategy for the prevention and control of salmonellosis.

Keywords: Bacillus subtilis; Salmonella Typhimurium; Immunity; Probiotic; Protection.

Fig. 1

Inhibitory effect of early colonization by Bacillus subtilis GX15 on the growth of pathogenic bacteria. Panels A, B, and C show the growth patterns of Salmonella Typhimurium SM022, Escherichia coli, and Staphylococcus aureus, respectively, following 24 h of pre-colonization by B. subtilis GX15 and a subsequent 24-hour co-culture with each pathogen. Panels D, E, and F represent the corresponding control groups, where each pathogenic bacterium was cultured independently without prior colonization by B. subtilis. The blue, green, yellow, and red arrows indicate S. Typhimurium SM022, E. coli, S. aureus, and B. subtilis GX15, respectively

Fig. 2

Inhibition of S. Typhimurium SM022 by B. subtilis GX15 in vitro. Results of different growth curves of S. Typhimurium alone and its co-culture with Bacillus subtilis GX15. The blue line represents the growth curve of Salmonella alone, and the red line represents the growth curve of Salmonella when co-cultured with Bacillus subtilis

Fig. 3

Effect of oral administration of B. subtilis GX15 on the severity of infection with S. Typhimurium in C57BL/6 mice. Panel A shows the effect of B. subtilis GX15 on body weight changes in mice infected with S. Typhimurium. Panels B and C depict the effects on the thymus index and spleen index, respectively. Error bars represent standard deviation (SD). Statistically significant differences are indicated as * (p ≤ 0.05) and *** (p ≤ 0.001), based on Tukey’s HSD test following one-way ANOVA. All experiments were performed in triplicate

Fig. 4

Effect of oral administration of B. subtilis GX15 on the cecum, spleen, and liver of C57BL/6 mice. Panel A shows representative liver histopathology sections from different experimental groups. Inflammatory cell infiltration is indicated by black circles. Panels B, C, and D display the bacterial load of S. Typhimurium SM022 in the liver, spleen, and cecum, respectively, comparing the B. subtilis prophylaxis group (BS + Sty) with the infected control group (NS + Sty). Error bars represent standard deviation (SD). Statistically significant differences are denoted as * (p ≤ 0.05) and ** (p ≤ 0.01), based on Tukey’s HSD test following one-way ANOVA. All experiments were performed in triplicate

Fig. 5

Antioxidant indices in mice pretreated with Bacillus subtilis GX15 or normal saline and subsequently infected with Salmonella Typhimurium SM022. C57BL/6 mice were treated for 10 days with B. subtilis GX15 (BS + Sty), normal saline followed by infection with S. Typhimurium SM022 (NS + Sty), or normal saline alone (CON). Panel A shows superoxide dismutase (SOD) levels, panel B displays total antioxidant capacity (T-AOC), and panel C presents glutathione peroxidase (GSH-PX) levels across the three groups. Error bars represent standard deviation (SD). Statistically significant differences are indicated as * (p ≤ 0.05) and **** (p ≤ 0.0001), based on Tukey’s HSD test following one-way ANOVA. All experiments were conducted in triplicate

Fig. 6

Effect of B. subtilis GX15 on immunoglobulins in C57BL/6 mice infected with S. Typhimurium SM022. The graph-A represents the IgG level in three groups. the graph-B represents the IgM level in three groups. the graph-C represents the IgA level in three groups.The error bars represent the standard deviation. Tukey's HSD test was used in the ANOVA. This experiment was performed in triplicate

Fig. 7

Bacillus subtilis GX15 modulates cytokine mRNA expression during Salmonella Typhimurium infection in C57BL/6 mice. Panels A–F show the relative mRNA expression levels of GM-CSF, IL-6, IL-10, TGF-β, TLR2, and IL-12p70, respectively, in small intestinal tissue. Error bars represent standard deviation (SD). Statistically significant differences are indicated as * (p ≤ 0.05) and *** (p ≤ 0.001), based on Tukey’s HSD test following one-way ANOVA. All experiments were performed in triplicate

Fig. 8

B. subtilis GX15 attenuated intestinal inflammation during S. Typhimurium SM022 infection. Panel A shows the villus length of the small intestine across the three experimental groups. Panel B presents the crypt depth, and Panel C displays the villus height-to-crypt depth ratio. Error bars represent standard deviation (SD). Statistically significant differences are indicated as * (p ≤ 0.05), ** (p ≤ 0.01), and *** (p ≤ 0.001), based on Tukey’s HSD test following one-way ANOVA. All experiments were performed in triplicate