Abstract
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.
Keywords: Upper respiratory tract microbiota, Streptococcus oralis (S. oralis), macrophages, mycobacteria
Highlight box.
Key findings
• Upper respiratory tract-derived Streptococcus oralis (S. oralis) obtained from close contacts of tuberculosis (TB) patients produced culture supernatants that inhibited the growth of Bacillus Calmette-Guérin, H37Rv, and Mycobacterium smegmatis, reduced intracellular mycobacterial burdens in THP-1-derived macrophages, and decreased bacterial loads in surrogate mouse infection models.
What is known, and what is new?
• Commensal microbiota can regulate respiratory immune homeostasis and influence host susceptibility to respiratory infections; however, the functional contribution of upper respiratory tract commensals to anti-TB defense remains poorly understood.
• This study identified S. oralis as a candidate functional commensal bacterium with anti-mycobacterial activity. Its secreted products appear to contain protease-sensitive, proteinaceous, or peptide-like bioactive components and are associated with reduced mTOR and p62 expression and increased LC3B-II abundance, suggesting activation of macrophage autophagy-related pathways.
What is the implication, and what should change now?
• These findings suggest that upper respiratory tract commensals and their secreted molecules may contribute to early anti-mycobacterial defense. Functional screening of respiratory microbiota should be further integrated into TB research, and future studies should identify the active molecules, confirm the causal relationship between autophagy activation and bacterial control, and validate the findings in primary macrophages and Mycobacterium tuberculosis infection models.
Introduction
Tuberculosis (TB), caused by infection with Mycobacterium tuberculosis (Mtb), is a chronic infectious disease and remains a major global public health threat (1,2). A substantial proportion of the global population is infected with or harbors Mtb, with most individuals remaining in a state of latent TB infection (3-5). Among individuals with latent infection, approximately 5–10% may progress to active TB when immune function declines or under other permissive conditions (3,4). Thus, protective host immunity is essential for restricting Mtb infection and maintaining long-term control of the pathogen (6-8).
Mtb primarily enters the host through the respiratory tract. During early infection, macrophages serve as both key effector cells mediating phagocytosis and pathogen clearance and host cells supporting intracellular survival and replication of Mtb (6,9,10). Mtb can impair macrophage bactericidal function by inhibiting phagosome maturation, interfering with phagosome-lysosome fusion, and modulating host cell autophagy (11,12). Therefore, enhancing macrophage-mediated antimicrobial responses, particularly autophagy-associated host defense, represents an important host-directed strategy for anti-TB intervention (12,13). In addition, the host cell microenvironment, macrophage lysosomal function, metabolic adaptation, and cellular heterogeneity can all influence Mtb control and treatment responses, suggesting that macrophage state is an important contextual factor when evaluating antimicrobial interventions (14-19).
In recent years, numerous microbiome studies have shown that commensal microbial communities not only help maintain host immune homeostasis but also influence susceptibility to respiratory infections and disease progression (20,21). Previous studies have largely focused on the remote regulation of the pulmonary immune microenvironment by the gut microbiota through the gut-lung axis (22-25). In contrast, the upper respiratory tract, as the initial portal through which Mtb enters the host, harbors complex and dynamic microbial communities. Local commensals and their secreted products may directly contribute to mucosal barrier function and anti-infective defense during early pathogen invasion (26-29). Evidence suggests that dysbiosis of the upper respiratory tract microbiota is closely associated with the development and progression of multiple respiratory infections (27,28). Streptococcus is a dominant commensal genus in the upper respiratory tract. It can influence pathogen colonization by producing bacteriocins and other bioactive metabolites, and may also participate in host immune regulation (30). However, the functional role of the upper respiratory tract microbiota in host anti-TB immunity remains unclear.
Previous animal-model and clinical studies have also shown that TB or nontuberculous mycobacterial disease can be accompanied by alterations in the structure of the respiratory microbiota (31-33). The upper respiratory tract or oropharyngeal microbiota has been associated with acute respiratory infections, the risk of lower respiratory tract infection, and local broad anti-infective states (34-36). The gut microbiota may also participate in TB or nontuberculous mycobacterial infection through modulation of inflammatory status and immune regulation (37-41).
Close contacts with long-term exposure to sputum-positive TB patients who nevertheless remain persistently tuberculin skin test (TST)/QuantiFERON-TB (QFT)-negative may possess relatively effective local or systemic anti-infective defense mechanisms that enable rapid and efficient elimination of incoming Mtb. Further research is required to determine whether the upper respiratory tract commensal microbiota and its products in this population contribute to defense during the early stage of Mtb invasion. Thus, the present study aimed to preliminarily investigate the anti-mycobacterial activity of microbial culture supernatants derived from throat swabs of these individuals, to isolate and identify candidate functional strains, and to characterize the active components and their effects on macrophage bactericidal function. In parallel, autophagy-related protein and mouse models of surrogate mycobacterial infection were analyzed to explore the potential mechanisms of action. The study findings may provide functional evidence for the involvement of upper respiratory tract commensals in host anti-mycobacterial defense and support the development of host-directed anti-TB strategies based on bioactive molecules derived from commensal bacteria. We present this article in accordance with the ARRIVE and MDAR reporting checklists (available at https://jtd.amegroups.com/article/view/10.21037/jtd-2026-2039/rc).
Methods
Study participants and ethics statement
The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study protocol was approved by the Ethics Committee of Shanghai Pulmonary Hospital, School of Medicine, Tongji University (approval No. K21-266), and all participants provided written informed consent. In total, the close contacts of 12 patients with TB were enrolled in the study. The enrolled participants had a mean age of 50.92±12.29 years (female/male ratio 7/5), a body mass index of 21.75±3.49 kg/m2, and all were negative for QFT-GIT (0/12) and human immunodeficiency virus (HIV). No active hepatitis B or C infection was present. The inclusion criteria were as follows: household members of sputum-positive TB patients; persistently negative TST and QFT Gold In-Tube results after at least 2 years of follow-up; and absence of clinical symptoms or imaging evidence of active TB. Throat swab samples were collected for subsequent microbial culture and microbiota analysis. All participants with HIV infection, immunodeficiency, diabetes, or other severe systemic diseases were excluded.
Bacterial strains, cell line, and culture conditions
The Mtb reference strain H37Rv (ATCC 27294), the attenuated Mycobacterium bovis Bacillus Calmette-Guérin (BCG) strain (ATCC 35733), and M. smegmatis (ATCC BAA-535) were maintained in our laboratory. The human monocytic leukemia cell line THP-1 (Cell Bank of the Chinese Academy of Sciences, SCSP-567) was routinely cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco, Waltham, MA, USA, #11875093) supplemented with 10% fetal bovine serum (Gibco, #10099141) and 1% penicillin-streptomycin (Gibco, #15140122). THP-1 cells were treated with 100 ng/mL phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich, Burlington, MA, USA, #P8139) for 24 h to induce differentiation into adherent macrophage-like cells before subsequent experiments.
Preparation of throat swab-derived microbial culture supernatants
Throat swab samples were enriched by static culture in antibiotic-free R10 medium at 37 ℃ for 16–18 h. Cultures were centrifuged at 10,000 ×g for 15 min at 4 ℃, and the supernatants were collected and filtered through 0.22-μm sterile membranes to obtain cell-free microbial culture supernatants, which were aliquoted and stored at −80 ℃ until use.
Evaluation of in vitro anti-mycobacterial activity
The antibacterial activity of culture supernatants was evaluated using in vitro growth inhibition assays and intracellular killing assays in macrophages. For the in vitro growth inhibition assay, BCG, H37Rv, or M. smegmatis was co-incubated with different concentrations of culture supernatant. After incubation at 37 ℃ for the indicated period, samples were serially diluted and plated on 7H10 agar containing OADC (oleic acid, albumin, dextrose, and catalase) for colony-forming unit (CFU) enumeration.
For intracellular killing assays, PMA-differentiated THP-1 macrophages were infected with BCG or H37Rv at a multiplicity of infection (MOI) of 10, or with M. smegmatis at a MOI of 1, for the indicated periods. Extracellular bacteria were removed by phosphate-buffered saline (PBS) washing, and complete medium containing different concentrations of culture supernatant was then added. At the end of incubation, cells were lysed with sodium dodecyl sulfate (SDS), and the lysates were serially diluted and plated for CFU enumeration, the CFU index was subsequently calculated by normalizing the raw counts to the control group. All experiments were performed independently three times, with three technical replicates in each experiment.
16S rRNA gene amplicon sequencing and microbiota analysis
Bacterial pellets obtained after enrichment culture of the throat swab samples were used for bacterial genomic DNA extraction, and the V3–V4 region of the 16S ribosomal ribonucleic acid (rRNA) gene was amplified and sequenced. Microbiota composition was subsequently analyzed, and relevant bioinformatics tools were used to predict the potential functional and phenotypic characteristics of the microbial communities.
Isolation and identification of functional strains
Monoclonal bacterial strains were isolated from the throat swab samples with the strongest activity using the plate streaking method on 7H10 agar plates. Single colonies with typical morphology were individually inoculated into brain heart infusion (BHI) liquid medium and cultured at 37 ℃ to the logarithmic growth phase. Cell-free culture supernatants were then collected and re-screened for anti-mycobacterial activity to identify the functional strain with the strongest inhibitory effect. Whole-genome sequencing was performed for the selected strain, and the sequencing data were assembled, genes were predicted, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotation was conducted. The raw sequencing reads have been deposited in the NCBI Sequence Read Archive (SRA) under BioProject PRJNA1503707.
Physicochemical characterization of the active components
Fractional ethanol precipitation was used to preliminarily separate active components in the culture supernatant. Absolute ethanol was added dropwise to the supernatant to final concentrations of 0%, 10%, 30%, and 60% (v/v). The anti-mycobacterial activity of each fraction was assessed as described above. To evaluate whether the active components were protease-sensitive, the culture supernatant was incubated with proteinase K at a final concentration of 100 μg/mL at 37 ℃ for 2 h. Phenylmethylsulfonyl fluoride was added to terminate the reaction, after which residual antibacterial activity was measured. A heat-inactivated proteinase K-treated control was included. All experiments were performed independently three times.
Western blot analysis of autophagy-related proteins
PMA-differentiated THP-1 macrophages were treated with different concentrations of Streptococcus oralis (S. oralis) culture supernatant for 12 h in the presence or absence of BCG infection (MOI =1 for 1 h). Cells were then harvested, and total protein was extracted. Western blotting was performed using antibodies against mammalian target of rapamycin (mTOR), p62, microtubule-associated protein 1A/1B-light chain 3B (LC3B), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). GAPDH served as the internal control, and chemiluminescent signals were developed and quantified by densitometry. Experiments were performed independently three times.
Mouse mycobacterial infection models and in vivo treatment
Animal experiments were performed under a project license (No. K22-143Z) granted by the Ethics Committee of Shanghai Pulmonary Hospital, School of Medicine, Tongji University, in compliance with the institutional guidelines for the care and use of animals. Female C57BL/6 mice [specific pathogen-free (SPF) grade] were obtained from GemPharmatech Co., Ltd. (Nanjing, China) and housed in an SPF animal facility at 22–25 °C and 50–60% relative humidity under a 12-hour light/dark cycle, with free access to food and water. All procedures were performed in accordance with animal welfare standards. Female C57BL/6 mice aged 6–8 weeks were randomly assigned to groups using a computergenerated random number table, with five mice in each group. The sample size was determined based on previous studies and experimental feasibility, without a formal power calculation. In the acute M. smegmatis infection model, mice received 200 μL of cell-free culture supernatant from the functional strain or BHI control by oral gavage once daily beginning 3 days before infection. After 3 consecutive days of gavage, mice were infected intravenously with M. smegmatis (1×106 CFU per mouse), continued to receive treatment for 2 days after infection, and were sacrificed on day 7. In the systemic BCG infection model, mice were infected intravenously with BCG (1×106 CFU per mouse). Beginning on day 3 after infection, mice received daily oral gavage for 7 consecutive days and were sacrificed on day 9. Lungs and spleens were aseptically collected, weighed, and homogenized. Tissue homogenates were serially diluted and plated on 7H10 agar plates, incubated at 37 ℃, and colonies were counted. CFU enumeration was performed by an investigator blinded to the group allocation. All mice were included in the final analysis, with no exclusions. Results are expressed as CFU.
Statistical analysis
All data are presented as the mean ± standard deviation. Statistical analyses and graphing were performed using GraphPad Prism version 8.0. Comparisons between two groups were performed using t-tests. Comparisons among multiple groups were performed using one-way or two-way analysis of variance. P<0.05 was considered statistically significant (*, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001). Data normality was assessed using the Shapiro-Wilk test before parametric analysis. All in vitro experiments were independently performed at least three times.
Results
Throat swab-derived microbial culture supernatants from TB close contacts exhibit anti-mycobacterial activity
To evaluate the anti-mycobacterial potential of soluble products derived from the upper respiratory tract microbiota, the in vitro inhibitory activity of microbial culture supernatants prepared from the throat swabs of 12 close contacts of patients with TB were first examined. In a cell-free culture system, pooled culture supernatants significantly inhibited BCG growth, and the inhibitory effect of the 50% supernatant group was stronger than that of the 10% supernatant group (Figure 1A). Further comparison of culture supernatants from different individuals revealed marked inter-individual variation, with samples 5 and 11 showing the most prominent inhibition of BCG growth (Figure 1B). Representative colony images were consistent with this inhibitory pattern (Figure 1C).
Figure 1.
Screening of the anti-mycobacterial activity of throat swab-derived microbial culture supernatants from TB close contacts. (A) Effects of pooled throat swab-derived microbial culture supernatants on the in vitro growth of BCG. (B) Effects of culture supernatants derived from 12 individual participants on the in vitro growth of BCG. The red boxes highlight samples 5 and 11, which showed the most prominent inhibitory activity against BCG growth among all 12 individuals tested. (C) Representative colony images showing the inhibitory effects of culture supernatants from different individuals on BCG growth. (D-F) PMA-differentiated THP-1 macrophages were infected with BCG (D), H37Rv (E), or M. smegmatis (F), and treated with culture supernatants at the indicated concentrations; intracellular viable bacterial loads were then measured. For the H37Rv infection shown in (E), the 50% supernatant concentration was not tested owing to limited volume of the pooled supernatant. Results are expressed as the CFU index and presented as the mean ± standard deviation. *, P<0.05; **, P<0.01; ****, P<0.0001; ns, not significant. BCG, Bacillus Calmette-Guérin; CFU, colony-forming unit; TB, tuberculosis.
The effects of culture supernatants on intracellular mycobacterial burden were then evaluated using a PMA-differentiated THP-1 macrophage infection model. Culture supernatant treatment significantly reduced viable intracellular burdens of BCG, H37Rv, and M. smegmatis in macrophages in a dose-dependent manner (Figure 1D-1F). For H37Rv, the 50% concentration was not tested with the pooled supernatant owing to limited volume; however, dose-dependent inhibition was confirmed with the strain 11-9 supernatant in subsequent experiments. These findings indicate that throat swab-derived microbial culture supernatants from close contacts of patients with TB can directly inhibit mycobacterial growth and reduce intracellular mycobacterial burden in a macrophage infection model, thereby providing an experimental basis for subsequent isolation and identification of functional strains.
Upper respiratory tract microbiota analysis and isolation and identification of candidate functional S. oralis strains
To characterize the upper respiratory tract microbiota of close contacts of patients with TB, 16S rRNA gene V3–V4 amplicon sequencing was performed on 12 throat swab samples. Taxonomic composition analysis showed that Streptococcus accounted for a relatively high proportion in most samples, with noticeable inter-individual variation in microbial community structure (Figure 2A,2B). Together with the previously observed differences in anti-mycobacterial activity among culture supernatants from different individuals, these findings suggest that Streptococcus may be associated with the inhibitory activity of certain samples and could serve as a candidate genus for subsequent functional strain isolation and validation.
Figure 2.
Composition and functional prediction analysis of the upper respiratory tract microbiota in TB close contacts. (A) Genus-level microbial community composition of 12 throat swab samples. (B) Composition of major taxa at lower taxonomic levels. (C) BugBase-predicted phenotypic features of the microbiota, including Gram-staining type, oxygen requirement, biofilm-forming potential, and potential pathogenicity. (D) Functional annotation heatmap based on the COG database, showing highly abundant functional categories across samples. Microbiota analysis was based on 16S rRNA gene V3–V4 amplicon sequencing of the throat swab samples. COG, Clusters of Orthologous Groups of proteins; TB, tuberculosis.
Further phenotypic and functional prediction analyses showed that the upper respiratory tract microbiota was mainly characterized by facultative anaerobic features and included both Gram-positive and Gram-negative bacteria (Figure 2C). Clusters of Orthologous Groups of proteins (COG)-based functional annotation suggested that the predicted functional potential of these microbial communities was primarily associated with substance transport, nutrient metabolism, and cell wall-related processes (Figure 2D). Based on the microbiota composition and culture supernatant activity results, Streptococcus was selected as the priority candidate genus for subsequent isolation and identification.
Plate streaking was then used to isolate 16 monoclonal strains from the highly active sample 11, and functional re-screening of their cell-free culture supernatants was performed using BCG as the target organism. The culture supernatants from different isolates exhibited clear differences in anti-mycobacterial activity, among which strain 11-9 showed relatively stable inhibitory effects (Figure 3A,3B). Further validation demonstrated that the culture supernatant of strain 11-9 not only inhibited BCG growth but also reduced viable intracellular burdens of BCG, H37Rv, and M. smegmatis in a THP-1-derived macrophage infection model (Figure 3C-3E), suggesting that it exerts direct growth-inhibitory activity and reduces intracellular mycobacterial burden.
Figure 3.
Isolation, identification, and anti-mycobacterial activity validation of a functional S. oralis strain. (A) Effects of candidate functional strain culture supernatants on the in vitro growth of BCG. (B) Effects of culture supernatants from monoclonal isolates obtained from sample 11 on the in vitro growth of BCG; the red box indicates strain 11-9, which was selected for subsequent experiments. (C-E) Changes in viable intracellular H37Rv, BCG, and M. smegmatis burdens in THP-1-derived macrophages after treatment with the culture supernatant of strain 11-9. Results are expressed as the CFU index and presented as the mean ± standard deviation. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. BCG, Bacillus Calmette-Guérin; CFU, colony-forming unit.
To determine the taxonomic identity of this functional strain, whole-genome sequencing of strain 11-9 was performed. Based on genome assembly and annotation, strain 11-9 was preliminarily identified as S. oralis (Table S1). KEGG functional annotation further showed that the genome of the strain was enriched in functional genes related to carbohydrate metabolism, amino acid metabolism, and membrane transport (Figure S1), suggesting a genetic basis for the production and transport of diverse bioactive metabolites. These results indicate that S. oralis is a candidate functional strain with anti-mycobacterial activity in this upper respiratory tract sample and provide a basis for further characterization of its active components and effects on host cells.
The anti-mycobacterial activity of S. oralis culture supernatant is associated with protease-sensitive components
To preliminarily characterize the physicochemical properties of the anti-mycobacterial active components in the S. oralis culture supernatant, the supernatant fractions were enriched by fractional ethanol precipitation, and the inhibitory activity of each fraction was assessed. In the cell-free culture system, the 30% and 60% ethanol-precipitated fractions both markedly inhibited the growth of BCG and M. smegmatis, with inhibitory effects approaching those of the unfractionated whole supernatant. Conversely, the water-treated control and the 10% ethanol fraction showed weak or minimal inhibitory activity (Figure 4A,4B). In the THP-1-derived macrophage intracellular infection model, the 30% and 60% ethanol-precipitated fractions likewise reduced viable intracellular BCG and M. smegmatis burdens (Figure 4C,4D). These results suggest that the anti-mycobacterial activity in the culture supernatant can be enriched in fractions precipitated at higher ethanol concentrations.
Figure 4.
Preliminary physicochemical characterization of anti-mycobacterial active components in S. oralis culture supernatant. (A,B) Effects of ethanol precipitation fractions on the in vitro growth of BCG and M. smegmatis. (C,D) Effects of different ethanol-precipitated fractions on viable intracellular BCG and M. smegmatis burdens in THP-1-derived macrophages. (E) Effects of culture supernatant on in vitro BCG growth after proteinase K treatment or heat-inactivated proteinase K control treatment. (F) Effects of culture supernatant on viable intracellular BCG burden in THP-1-derived macrophages after proteinase K treatment or heat-inactivated proteinase K control treatment. Results are expressed as the CFU index and presented as the mean ± standard deviation; *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001; ns, not significant. BCG, Bacillus Calmette-Guérin; CFU, colony-forming unit.
The culture supernatant was further digested with proteinase K. Proteinase K treatment markedly attenuated the direct inhibitory activity of the culture supernatant against BCG and also significantly reduced its ability to lower BCG burden in THP-1-derived macrophages. Conversely, the heat-inactivated proteinase K-treated control retained antibacterial activity similar to that of the untreated supernatant (Figure 4E,4F). These findings indicate that the anti-mycobacterial activity in the S. oralis culture supernatant is sensitive to protease treatment, suggesting that the main effector components may be proteins or peptides.
S. oralis culture supernatant reduces intracellular mycobacterial burden and is accompanied by changes in autophagy-related proteins
To explore the potential cellular mechanisms by which S. oralis culture supernatant reduces intracellular mycobacterial burden in macrophages, changes in the expression of autophagy-related proteins were examined. THP-1-derived macrophages were treated with different concentrations of S. oralis culture supernatant under uninfected or BCG-infected conditions, followed by Western blot analysis of mTOR, p62, and LC3B expression.
Under both the uninfected and BCG-infected conditions, treatment with 50% culture supernatant reduced mTOR and p62 protein levels in macrophages and significantly increased LC3B-II expression (Figure 5). These changes were more pronounced in the 50% culture supernatant group than in the 10% culture supernatant group, suggesting a degree of concentration dependence. Decreased p62 levels and increased LC3B-II levels are important indicators of changes in autophagy-related processes, whereas reduced mTOR signaling is commonly associated with the regulation of autophagy-related pathways. These results suggest that treatment with S. oralis culture supernatant is associated with alterations in the autophagy-related protein expression profile of THP-1-derived macrophages, which may contribute to its ability to reduce the intracellular mycobacterial burden.
Figure 5.
Effect of S. oralis culture supernatant on autophagy-related protein expression in macrophages. (A) Western blot analysis of mTOR, p62, LC3B-I/II, and GAPDH protein levels in THP-1-derived macrophages treated with different concentrations of the S. oralis culture supernatant under uninfected or BCG-infected conditions. (B-D) Densitometric quantification of the relative expression levels of mTOR, p62, and LC3B-II, respectively. GAPDH was used as the internal control. Data are presented as the mean ± standard deviation. *, P<0.05; **, P<0.01; ns, not significant. BCG, Bacillus Calmette-Guérin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
Oral streptococcal culture supernatant reduces tissue bacterial burden in mycobacteria-infected mouse models
To further evaluate the anti-mycobacterial activity of oral streptococcal culture supernatant in vivo, validation was performed using an acute M. smegmatis infection model and a systemic BCG infection model. In the acute M. smegmatis infection model, C57BL/6 mice were orally administered the culture supernatant of strain 11-9 according to the experimental protocol and subsequently infected with M. smegmatis via tail vein injection. The viable bacterial burden in lung tissues was subsequently assessed (Figure 6A). The results showed that, compared with the medium control group, mice treated with the culture supernatant of strain 11-9 exhibited a marked reduction in the viable M. smegmatis load in lung tissues (Figure 6B).
Figure 6.
In vivo antibacterial activity of S. oralis culture supernatant in mouse surrogate mycobacterial infection models. (A) Schematic workflow of the acute M. smegmatis infection model. C57BL/6 mice received culture supernatant from strain 11-9 by oral gavage, were infected intravenously with M. smegmatis, and were assessed for lung bacterial burden at the experimental endpoint. (B) Viable M. smegmatis burdens in lung tissue from each group. (C) Schematic workflow of the systemic BCG infection model. Mice were intravenously infected with BCG, treated by oral gavage with culture supernatant from strain 11-9 beginning on day 3 after infection, and assessed for tissue bacterial burden at the experimental endpoint. (D,E) Viable BCG burdens in the lungs and spleens of mice in each group. Results are expressed as the CFU index and presented as the mean ± standard deviation. *, P<0.05; **, P<0.01. BCG, Bacillus Calmette-Guérin; CFU, colony-forming unit.
In the systemic BCG infection model, mice were infected with BCG via tail vein injection and then orally administered the culture supernatant of strain 11-9 daily beginning on day 3 post-infection. Bacterial loads in the lungs and spleens were measured at the experimental endpoint (Figure 6C). The results showed that, compared with the medium control group, treatment with the culture supernatants of this strain reduced the BCG burden in lung tissues (Figure 6D) and showed a trend toward a reduced BCG burden in the spleen (Figure 6E). During the observation period, no deaths, obvious body weight loss, or abnormal behaviors were observed in any group.
Together, these results suggest that oral streptococcal culture supernatant exhibits in vivo antibacterial activity in mycobacteria-infected mouse models, providing experimental evidence for further validation of its effects in an Mtb infection model that more closely resembles the pathological process of TB.
Discussion
This study functionally explored the anti-mycobacterial role of upper respiratory tract commensal microbiota in close contacts of patients with TB. We found that microbial culture supernatants derived from a subset of throat swab samples exhibited anti-mycobacterial activity, and a candidate functional strain of S. oralis was isolated from a sample with strong inhibitory activity. The culture supernatant from this functional strain not only inhibited mycobacterial growth in a cell-free system but also reduced viable intracellular burdens of BCG, H37Rv, and M. smegmatis in THP-1-derived macrophages, indicating its potential to exert both direct growth-inhibitory effects and enhanced intracellular antimicrobial activity.
Unlike previous studies that have primarily focused on changes in respiratory microbiota composition, the present study further validates the anti-mycobacterial activity of products derived from upper respiratory tract commensals at a functional level. The upper respiratory tract is the initial portal through which Mtb enters the host, and local commensals and their secreted products may contribute to early pathogen colonization resistance and mucosal defense (26-29).
In this study, S. oralis was screened and identified from a sample with strong inhibitory activity, providing experimental evidence for the involvement of upper respiratory tract commensals in anti-mycobacterial defense. This finding is consistent with observations of airway microbiota dysbiosis in animal models of TB and respiratory microbiome alterations associated with nontuberculous mycobacterial disease (31-33). In addition, host microbial homeostasis may be influenced by regulation from myeloid cells such as macrophages, providing a biological basis for the effects of commensal-derived products on macrophage function (30).
Further analysis showed that the antibacterial activity of the S. oralis culture supernatant was enriched in the 30–60% ethanol-precipitated fractions and was sensitive to proteinase K treatment, suggesting that proteinaceous or peptide-like molecules may represent one of the main classes of active components. Meanwhile, culture supernatant treatment decreased mTOR and p62 levels and increased LC3B-II expression in macrophages, indicating that autophagy-related processes may contribute to the reduction of the intracellular mycobacterial burden. Given that impaired lysosomal function can increase susceptibility to TB and that macrophage differentiation, metabolic state, and heterogeneity influence the course of Mtb infection, the autophagy-related protein changes observed in this study are mechanistically plausible (15,17).
In vivo experiments showed that S. oralis culture supernatant reduced bacterial burdens in the lungs or spleen in an acute M. smegmatis infection model and a systemic BCG infection model, indicating that it also possesses anti-mycobacterial potential in vivo. However, this study had several limitations. First, the number of participants was small, and parallel comparisons with upper respiratory tract samples from healthy individuals, individuals with latent infection, and patients with active TB were lacking; therefore, it cannot yet be concluded that this phenomenon represents a protective mechanism unique to close contacts of patients with TB. Second, the precise molecular identity of the active components remains unresolved. Third, the relationship between autophagy-related changes and the antibacterial effect remains largely correlative, and additional experiments are required to establish causality. Further, this study used M. smegmatis and BCG surrogate infection models; thus, future research should seek to confirm the findings in Mtb infection models. Because the host cell environment itself can influence the effects of antimicrobial interventions, the activity of the culture supernatant should also be validated in macrophages from different sources, alveolar macrophages, and primary cell models (14).
In summary, this study identified a candidate functional S. oralis strain from upper respiratory tract samples of close contacts of patients with TB and found that its culture supernatant inhibits mycobacterial growth and reduces bacterial burden in macrophages. This effect may be associated with protease-sensitive active components and changes in autophagy-related proteins. Although we identified S. oralis as a candidate functional strain, a thorough evaluation of its pathogenicity-related genes, antibiotic resistance determinants, and mobile genetic elements is warranted before considering probiotic use. These analyses are currently underway and will be presented in a subsequent publication. The study provides functional evidence that upper respiratory tract commensals participate in host anti-mycobacterial defense and offers new research directions for developing commensal-derived bioactive molecules as host-directed anti-TB strategies.
Conclusions
In summary, this study identifies S. oralis from the upper respiratory tract of TB close contacts as a commensal strain with anti-mycobacterial activity. Its culture supernatant inhibits mycobacterial growth, reduces intracellular bacterial burden in macrophages, and decreases tissue bacterial loads in surrogate mouse infection models. The activity is associated with protease-sensitive components and altered autophagy-related protein expression, suggesting that commensal-derived products may contribute to early host defense against mycobacteria. These findings provide functional evidence for the involvement of respiratory commensals in anti-TB immunity and support further investigation into the active molecules and their potential as hostdirected therapeutic agents.
Supplementary
The article’s supplementary files as
Acknowledgments
None.
Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Ethics Committee of Shanghai Pulmonary Hospital, School of Medicine, Tongji University (approval No. K21-266), and written informed consent was obtained from all participants. Animal experiments were performed under a project license (No. K22-143Z) granted by the Ethics Committee of Shanghai Pulmonary Hospital, School of Medicine, Tongji University, in compliance with the institutional guidelines for the care and use of animals.
Reporting Checklist: The authors have completed the ARRIVE and MDAR reporting checklists. Available at https://jtd.amegroups.com/article/view/10.21037/jtd-2026-2039/rc
Funding: This project was supported by the Sanfeng Talent Program of Shanghai Pulmonary Hospital.
Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://jtd.amegroups.com/article/view/10.21037/jtd-2026-2039/coif). All authors report that the present work was supported by the Sanfeng Talent Program of Shanghai Pulmonary Hospital (funding paid to the institution). The authors have no other conflicts of interest to declare.
(English Language Editor: L. Huleatt)
Data Sharing Statement
Available at https://jtd.amegroups.com/article/view/10.21037/jtd-2026-2039/dss
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