This is the first study to unravel links between LTBI status, host immunity, and gut microbiome dysbiosis in patients with pDM. We demonstrated the following: (1) pDM patients with LTBI have a markedly different gut microbiome to individuals without LTBI; they had enriched Bacteroides, Alistipes, and Blautia, but depleted Prevotella_9, Streptococcus, and Actinomyces. (2) The proposed microbiota-based model involving the 6 most differential genera in gut microbiota had favorable performance with an accuracy of 0.872 in predicting LTBI status among patients with pDM. (3) The 6 discriminatory genera were correlated with a decrease in Th1/Th17 cell– mediated inflammatory cytokines and indicated to involve in depletion of immune, metabolism, and replication pathways.
Interferon gamma release assays (IGRAs), based on the detection of IFN-γ responses of peripheral lymphocytes against Mtb-specific antigens, are the current standard diagnostic test for LTBI. However, they have a low sensitivity in patients with DM, most likely because of the attenuation of IFN-γ release [21, 22]. Additionally, the results of sequential IGRAs cannot reflect dynamic changes in Mtb infection and identify subclinical TB . A new diagnostic platform incorporating various diagnostic modalities for LTBI is necessary to guide point-of-care
management and the timing of treatment initiation.
Studies have demonstrated that compared with healthy individuals, the gut microbiota of patients with DM have a lower relative abundance of genera Bacteroides , Alistepes  and Blautia  and a higher relative abundance of Actinomyces  and Prevotella . Unexpectedly, the current study demonstrated that the gut microbiota composition of DM patients with LTBI was turned out to be similar to healthy individuals, which implies that LTBI status may be associated with the dysbiotic microbiota of patients with pDM. Furthermore, growing evidence indicates that the disruption of gut microbiome equilibrium can contribute to changes in TB stage [14, 28, 29], which could provide clinical utility for LTBI diagnosis beyond conventional IGRAs.
The most differential bacterial taxa between pDM patients with and without LTBI identified by the current prediction model have previously been associated with TB susceptibility based on the regulation of Th1/Th17 immune responses and inflammation [14, 28–31]. Although the role of Streptococcus and Actinomyces had not previously been identified in patients with TB, patients with active TB have a lower phylogenetic diversity and a significantly lower abundance of short chain fatty acid (SCFA)–producing bacteria such as Bacteroidetes, Alistepes, and Prevotella compared with healthy individuals [32–34]. Conversely, the unique gut microbiome features with high abundance of SCFA producers (Bacteroides and Alistepes) in current pDM patients with LTBI may enhance TB susceptibility by suppressing B cells and CD4 + and CD8 + lymphocytes, reducing the production of TB-induced IFN-γ and IL-17, increasing Foxp1 expression [14, 28, 30, 31] and elevating the number of T regulatory cells in peripheral blood . Blautia was reported to be more abundant in patients with TB than in healthy household contacts; transcriptome analysis indicated that this abundance may be related to inflammation-modulating pathways . Paradoxically, Prevotella had diverse immunomodulatory properties in different TB stages; it was positively correlated with CD4 + cell counts in patients with newly diagnosed TB but negatively correlation with such cell counts in patients with TB recurrence . Further mechanistic studies are required to confirm the immunomodulatory effect of each relevant taxon.
In accordance with the findings in previous studies [9, 36], this study revealed significantly lower plasma levels of TNF-α and IL-17 in DM patients with LTBI than non-LTBI counterparts, suggesting increased TB susceptibility through the decreasing phagocytic ability of macrophages, interference with granuloma formation  and inhibiting Mtb-specific memory responses . The significant correlation between Th1 -and Th17-related cytokines and the 6 most differentially expressed taxa of gut microbiota indicates that gut microbiota have an immunomodulatory effect on Mtb infection. Furthermore, the significant downregulation of gene expression in immune, metabolism, and replication pathways in the LTBI group was in accordance with the evasion of immune surveillance through the suppression of host immunity, a reduction in energy expenditure, and the attainment of an intracellularly nonreplicant dormancy status by Mtb . Taken together, the findings of the current study provide substantive evidence that gut microbiota composition may predict alterations in the immune status of patients with pDM after Mtb infection. Furthermore, the findings could provide a first step toward host-directed immunomodulatory therapy through the precise tuning of the enteric microbiome to enhance host immunity against Mtb infection.
This study had several limitations. First, this cross-sectional study could not determine causality or the mechanisms behind the effect of gut microbiota alterations on host immunity during various stages of Mtb infection. Second, using 16s rRNA sequencing rather than shotgun metagenomics may interfere the taxonomic and functional resolution of microbiomes owing to the inadequacy of gene-related information obtained from strains. Third, current study did not reveal the influence of microbiome-derived metabolite alterations on TB pathogenesis. Further metabolomics studies should be conducted to verify this influence. Forth, ethnicity and regional variations among individuals can alter the precision of microbiome-based diagnostics. An external validation of the proposed prediction model is necessary.