Oral delivery of recombinant Bacillus subtilis expressing mSEB reduces intestinal Staphylococcus aureus colonization.
BACKGROUND S. aureus intestinal colonization elevates infection risk, underscoring decolonization as a key prevention target. SEB is a key target for the development of neutralizing antibodies against toxins to prevent and treat S. aureus infection. B. subtilis is used as a probiotic for human health. OBJECTIVE To investigate the efficacy of recombinant mSEB B. subtilis spores for reducing S. aureus intestinal colonization in mouse. METHODS Mice were orally immunized with recombinant mSEB spores three times a week for three weeks. After immunization, mice were challenged via oral gavage with 1 × 109 CFU of S. aureus (ATCC 14458). Fecal specific IgA and serum IgG1 and IgG2a were analyzed by ELISA. Peritoneal macrophages were isolated for qRT-PCR analysis of TNF-α, IL-6 and IL-1β mRNA expression. Colon contents samples underwent 16S rRNA sequencing for microbiota profiling. Intestinal RNA was extracted for transcriptome sequencing (RNA-seq), and differential pathways were analyzed using KEGG enrichment. Body weight and fecal viable S. aureus burden were monitored. RESULTS Oral mSEB spores correlated with elevated systemic and mucosal SEB-specific IgG1, IgG2a and fecal sIgA (P < 0.01). After S. aureus challenge, peritoneal macrophages from mSEB mice showed lower pro-inflammatory TNF-α, IL-6 and IL-1β transcripts, consistent with attenuated systemic inflammation. mSEB immunization also altered gut microbiota and increased the relative abundance of Barnesiella. Intestinal RNA-seq identified enriched antigen presentation and B cell receptor signaling gene sets in the mSEB group. On day 3 post-challenge, fecal S. aureus loads were 24.20 ± 3.967 × 104 CFU (Control), 8.081 ± 3.614 × 104 CFU (CotC) and 3.6 ± 1.030 × 104 CFU (mSEB), showing a pathogen-clearing phenotype linked to mSEB treatment. CONCLUSION Immunization with mSEB-displaying Bacillus subtilis spores correlates with SEB-specific mucosal and systemic antibody responses, blunted systemic inflammation, modified gut microbiota, and reduced intestinal S. aureus burdens post-challenge. The causal mechanisms underlying these changes remain to be clarified.