Upper respiratory tract commensal Streptococcus oralis inhibits mycobacterial growth by producing bioactive molecules and inducing macrophage autophagy

Background: Tuberculosis (TB) is caused by Mycobacterium tuberculosis (Mtb) and remains a major global health challenge. Although host immunity is critical for controlling infection, the role of upper respiratory tract commensal microbiota in anti-mycobacterial defense remains unclear. This study investigated whether commensal bacteria from close contacts of patients with TB, who remained persistently tuberculin skin test- (TST) and QuantiFERON-TB (QFT) Gold-negative after long-term exposure, produce factors capable of inhibiting mycobacterial growth.

Methods: Throat swabs were collected from close contacts of patients with TB and subjected to ex vivo enrichment culture. Cell-free microbial culture supernatants were tested for activity against Bacillus Calmette-Guérin (BCG), H37Rv, and Mycobacterium smegmatis (M. smegmatis) in cell-free culture and THP-1-derived macrophage infection models. Microbiota composition was analyzed by 16S ribosomal ribonucleic acid (rRNA) gene amplicon sequencing, and functional strains were isolated and identified by whole-genome sequencing. The physicochemical properties of active components were examined using ethanol precipitation and proteinase K digestion. Autophagy-related proteins were assessed by Western blotting, and in vivo activity was evaluated in mouse models of M. smegmatis and BCG infection.

Results: Supernatants from selected throat swab cultures significantly inhibited mycobacterial growth and reduced intracellular bacterial burdens in macrophages. Microbiota analysis and functional screening identified Streptococcus oralis (S. oralis) as a candidate anti-mycobacterial commensal. The culture supernatant of an isolated S. oralis strain inhibited BCG, H37Rv, and M. smegmatis, and decreased intracellular mycobacterial survival. Its activity was enriched in ethanol-precipitated fractions and was markedly reduced after proteinase K digestion, suggesting proteinaceous or peptide-like active components. Treatment with S. oralis supernatant reduced mTOR and p62 expression and increased LC3B-II abundance, consistent with activation of autophagy-related pathways. Oral administration of the supernatant reduced bacterial loads in mouse infection models.

Conclusions: Upper respiratory tract-derived S. oralis may contribute to anti-mycobacterial defense by producing protease-sensitive bioactive molecules and modulating macrophage autophagy-related pathways. These findings support further investigation of respiratory commensal-derived products as potential host-directed anti-TB strategies.