Gut microbiota profile in newly diagnosed pulmonary tuberculosis patients: an exploratory pilot study in southern India

Tuberculosis (TB) is among the most widespread infectious illnesses, inflicting considerable distress globally. Mycobacterium tuberculosis (Mtb) is the sole etiological agent of TB, primarily targeting the lungs, referred to as pulmonary tuberculosis (PTB). However, it may also impact other organs. As per the Global TB Report 2024, the cases of TB were 10.8 million in 2023 [1]. In 2023, a global total of 8.2 million individuals were newly diagnosed with TB, an increase from 7.5 million in 2022 [1]. Five nations represented 56% of the worldwide TB cases [1]. Among these, India contributes 26%, followed by Indonesia (10%), China (6.8%), the Philippines (6.8%), and Pakistan (6.3%). In 2023, 1.09 million fatalities worldwide were formally attributed to TB and recorded. Among HIV-negative persons, India alone accounted for 29% of the worldwide total of TB-caused deaths [1].

The gut microbiota comprises many microbes, including prokaryotes, archaea, viruses, and microbial eukaryotes [2]. The gut and lung axis works through bidirectional microbial communication between the gut and lungs. The link between PTB and gut microbiota dysbiosis is an emerging research area. The pleiotropic effect of gut microbiota has illuminated the possibility of its influence on TB [3]. Unlike genetic epidemiology, the microbiome is far more amenable to modification than the human genome, as several host and environmental factors can influence and reshape it. Thereby enabling its alteration to be harnessed for a wide range of therapeutic applications [4]. Advancements in next-generation sequencing technologies have enhanced our understanding of the gut microbiota in TB [5, 6].

Previous studies have reported that probiotics may help alleviate anti-tuberculosis treatment (ATT)-induced adverse drug reactions, particularly during the intensive phase of therapy, making them of clinical interest in TB management [7, 8]. In a subset of enrolled patients in our study, probiotic supplements were prescribed for good gut health during (ATT) based on the treating physician’s’ discretion and hospital formulary availability. This allowed for an exploratory evaluation of how probiotics supplementation might influence gut microbiota in PTB patients undergoing ATT. In this study, we aimed to characterize the gut microbiota of PTB patients from the southern Indian population in terms of microbial diversity, marker taxa identification, and inferred microbial functionality.

This study provides insights into the gut microbial changes linked to PTB within the southern Indian demographic. The diversity of gut bacterial microbiota decreased more in the naïve PTB patient group than in the HC group. Furthermore, beta diversity demonstrated notable differences in microbial community composition between PTB patients (PTB_before_ATT) and HC (p = 0.001), as evidenced by distinct clustering in PCoA plots. These findings are corroborating earlier research [17, 18]. Ding et al. 2022 observed a significant reduction in gut microbial diversity among PTB patients compared to HC [17]. Similarly, Wang et al. 2021 reported distinct clustering patterns in PTB patients, reflecting substantial shifts in the microbial community structure [18].

Our study findings from comparing the PTB_after_ATT group with the HC group revealed that the conventional treatment by ATT further exacerbates microbial dysbiosis in PTB patients than in naïve PTB patients. This suggests that while ATT is essential for managing PTB, it may worsen gut microbial imbalances. The resilience of dysbiosis, as evidenced by persistent differences from HCs, underscores the need for adjunctive interventions to restore microbial health during treatment. Notably, in our study, samples from PTB patients were collected after the ATT exposure time was limited to two months (intensive phase). This indicates that ATT quickly and substantially affects gut microbial depletion. Wipperman et al. 2017 demonstrated that dysbiosis induced by ATT persists for at least 14 months, implying ATT’s long-term impact on the gut microbial community [19]. Similarly, Séraphin et al. 2023 concluded that rifamycin-based TB preventive therapy also induces depletion in microbial diversity [20]. The alpha diversity indices increased two months after rifamycin treatment, yet failed to achieve the baseline level, implying the incomplete recovery in microbial community diversity. Furthermore, the potential impact of mono and combined drug therapy for TB patients could be considered, as different drug regimens may have varying effects on the degree of gut microbiota dysbiosis.

The subgroup analysis from paired samples comparison of PTB patients before and after receiving ATT did not yield any significance in microbial diversity, yet showed marker taxa in differential abundance analysis. Nevertheless, it is crucial to recognize that our findings result from a minimal sample size of PTB patients after ATT initiation, which restricts the statistical generalizability of these findings. Despite this limitation, we observed significant changes in taxa from differential abundance analysis at the genus level, emphasizing specific microbial modifications linked to ATT.

Interestingly, the supplementation of probiotics alongside ATT demonstrated a modest but notable improvement in microbial diversity. In the PTB_after_ATT_Probiotics group, alpha diversity metrics showed mild increases compared to the PTB_after_ATT group. However, this improvement was not sufficient to achieve statistical equivalence with HCs. Yet, the observed trends suggest a beneficial modulation of gut microbiota by probiotics. Probiotic supplementation during ATT is likely to modulate the disruption of the gut microbiota, which ATT alone does not address. It emphasizes how probiotics may maintain or recover microbial diversity in PTB patients during the long-term ATT. John et al. 2024 concluded that the probiotic may minimize the disruption of antibiotic treatment on the gut microbiome by preserving microbial diversity and even reducing the abundance of antimicrobial resistance genes [21]. Subgroup analyses provided further insights into the role of probiotics. Paired sample comparisons of PTB patients before and after ATT, receiving probiotics, revealed significant improvements in alpha diversity metrics (Chao1, Observed taxa, Fisher diversity, rarity of low-abundance taxa). However, beta diversity metrics in these paired before and after analyses did not exhibit significant shifts, indicating that probiotics may influence specific microbial components rather than driving broader compositional changes. It is important to note that in this before-and-after ATT probiotics pairwise comparison, rarefaction was not performed before diversity analysis to retain all matched-pairs samples. While this approach preserves within-sample pairing for probiotics supplementation, it may limit the direct comparability of diversity metrics with other rarefied group comparisons.

Healthy human gut microbiota is dominated by the phyla Firmicutes and Bacteroidota [22], consistent with our findings in the HC group. Both active PTB disease and ATT are associated with gut microbiota dysbiosis [23‐25]. Similarly, our study findings revealed differences in gut microbiota composition in PTB_before_ATT and PTB_after_ATT with respect to HC, suggesting a dysbiotic state.

Our study findings identified distinct microbial signatures across groups. In comparisons between HC and PTB_before_ATT, including Faecalibacterium, Roseburia, Agathobacter, Coprococcus, Ruminococcus, and Lachnospiraceae NK4A136 group, were significantly deregulated in PTB_before_ATT. These genera are known as Short-Chain Fatty Acids (SCFAs) producers [26‐31]. SCFAs, specifically acetate, propionate, and butyrate, are the primary metabolites generated by the gut microbiota. SCFAs have a variety of functions such as anti-inflammatory, immune regulations, etc [32]. Therefore, the depletion of SCFAs-producing gut commensals may contribute to immune dysregulation and systemic inflammation in PTB.

The genus Erysipelatoclostridium was abundant during the ATT, as reported by Wipperman et al., 2017, and Hu et al., 2019 [19, 33]. Erysipelatoclostridium is enriched during inflammatory and immune-reducing conditions, such as in TB patients [34]. These findings are consistent with our study findings, as we observed the following ATT in the PTB_after_ATT group. Furthermore, we detected the genus Erysipelatoclostridium in the PTB_after_ATT_Probiotics group, suggesting that this genus has persisted even with short-term probiotics supplementation. As per Wipperman et al. 2017 [19], the genus Erysipelatoclostridium persisted for three months following ATT. This indicates a long-term microbial shift induced by treatment. A short-term probiotics supplementation in our study may have been insufficient to restore microbial equilibrium or eliminate persistent taxa such as Erysipelatoclostridium. The marker genus Erysipelatoclostridium suggests continued dysbiosis or a transitional microbial state.

The marker genera, such as Clostridium sensu stricto 2, Terrisporobacter, Eubacterium, and Paraclostridium, were identified from paired samples comparison of PTB patients before and after receiving ATT. Furthermore, these genera are butyrate producers, which were identified to be depleted post-ATT [35‐38], similar to our study findings. Genus Clostridium sensu stricto is generally considered the true Clostridium genus [39]. Some species within Clostridium sensu stricto can be pathogenic, while others are considered beneficial. However, most species are considered harmless and responsible for SCFAs production [39]. These Clostridium sensu stricto species, as marker genera for microbial health, could be valuable targets for future studies.

In this study, microbial metagenomic inference was conducted to predict functional pathways that distinguish HC from PTB patients. Our findings revealed a deregulation of functional pathways in PTB patients following ATT, suggesting shifts in gut microbiota function associated with the treatment. However, probiotics supplementation during ATT resulted in more active metabolic pathways than ATT alone. Pathways enriched in HC are primarily involved in biosynthesis and energy metabolism, suggesting that healthy individuals’ gut microbiota is functionally balanced, supporting normal gut and host metabolism. The inferred pathways in PTB_before_ATT patients indicate significant dysbiosis with a shift towards catabolic and degradation pathways, which could be adapting to a pathological or nutrient-deprived environment. Menaquinone (Vitamin K2) biosynthesis is crucial in bacterial respiration, particularly in Gram-positive bacteria. Certain bacteria, including those in the gut, rely on menaquinone for electron transport in aerobic or anaerobic respiration. Respiration occurs in the cell membrane of prokaryotic cells [40]. An increase in Menaquinone biosynthesis pathways in PTB_after_ATT patients could indicate a microbial shift towards metabolic pathways that support native gut commensal bacterial survival and energy production in response to production imbalance during the treatment period. The before- and -after comparison of ATT_probiotics revealed enrichment in the L-methionine biosynthesis pathway, indicating the possible influence on gut microbiota functionality due to probiotics supplementation during ATT.

The current commercially available probiotics supplements can provide metabolically and ecologically beneficial effects, implying dynamic effects. These are not meant for gut colonization. These commercially available probiotic strains tend to pass through the digestive system and are usually flushed out within a few days [4]. For TB patients undergoing ATT, which has a minimum duration of six months, supplementation with probiotics for just one or two months may have a limited impact. Probiotic supplementation may not dynamically change the gut microbial community by displacing the native commensal microbes. Instead, it may only maintain the gut microbiota stability for eubiosis, protecting from the exacerbation of dysbiosis during ATT.

Our study only employed a 16 S amplicon sequencing approach for studying metataxonomics, which gave us the landscape of microbial community diversity and taxonomy. Future studies may focus on integrative multi-omics approaches such as metagenomics to capture the functional gene repertoire, metatranscriptomics to understand microbial transcriptional activity, and metabolomics to profile the metabolite outputs of microbial communities. Such approaches would facilitate a more profound exploration of the development of precision microbiome-based interventions.

Conventional probiotics are designed to benefit overall gut health. They are used for common issues like digestive discomfort, immune support, or general microbiome balance in the “one size fits all” concept. However, next-generation probiotics, defined as “live microorganisms identified based on comparative microbiota analyses that, when administered in adequate amounts, confer a health benefit on the host”, are more promising, with the potential for customized probiotic treatments [41, 42]. With a precision therapeutic approach, interventions like next-generation probiotics and prebiotics can be tailored to the local environment and the microbiome traits of a person or population [41, 42].

The follow-up duration in our study was limited to less than two months, which may not be sufficient to fully capture the long-term dynamics of the gut microbiota in PTB patients. Additionally, the relatively small sample size, particularly in the longitudinal comparison for subgroup analysis (n = 4 for PTB_after_ATT and n = 5 for PTB_After_ATT_Probiotic groups, respectively), substantially limits the statistical power and generalizability of the findings. Furthermore, we did not collect detailed information on participants’ diet or lifestyle factors, which could have significantly influenced gut microbiota composition. The absence of this data limits the ability to fully contextualize the observed microbial changes. While our study findings provide initial insights into the potential influence of probiotics, further studies with larger cohorts and extended follow-up are required to validate these results. Such studies are also needed to characterize the long-term impacts of ATT and probiotics on gut microbial communities, especially those taxes involved in SCFA production.

The study identified distinct gut microbial features in PTB patients in the south Indian population. The gut microbial diversity depletion was found among PTB patients, and this was further exacerbated during ATT. The supplementation of probiotics alongside ATT showed a trend toward restoring microbial diversity to higher levels than ATT alone. However, this improvement did not reach statistical significance. Moreover, our analysis revealed a notable depletion in the predicted metabolic pathways in PTB patients undergoing ATT, with biosynthesis pathways mainly affected. Furthermore, the study identified distinct microbial taxa in PTB patients before ATT initiation and during ATT, which could serve as potential biomarkers, highlighting opportunities for future research to explore targeted microbiome-based interventions to mitigate treatment-associated dysbiosis. Nevertheless, these findings warrant careful understanding due to the very small sample size in the follow-up groups, which limits generalizability. Larger, well-controlled studies are needed to validate and expand upon these preliminary results.