The human gut microbiome is critical for the development of the host immune system and tissue homeostasis [28]. Aside the numerous beneficial roles of the microbiome within an individual, its role in the development of metabolic and infectious diseases in an altered (dysbiotic) state has been documented [29]. In this study we explored the gut microbiome dynamics in TB patients with or without HIV or diabetes comorbidities before and after intensive phase anti-TB therapy. We found that the microbiome of the TB patients was enriched with inflammatory related microbial organisms with a general increase in these organisms after at 2 months HRZE intervention.
Several studies have reported a decreased gut microbiome diversity in TB patients [30–33], similar to findings in our study which shows a significantly lower gut microbial diversity in TB patients before HRZE intervention compared to healthy controls (Fig. 3a, 3b, 3c). This reduced alpha diversity persisted after 2 months of HRZE intervention especially in the comorbid cases (Fig. 4a, 4b). This could be indicative that in addition to TB, compositional shifts could be occurring in a TB-comorbidity specific manner.
Firmicutes are known to be a predominant phylum in the human gut [34–36]. Likewise in this study, the most abundant phylum in both healthy controls (76.3%) and all TB patients (> 65.8%) was the phylum Firmicutes (Supplementary Fig. 1). Several bacteria groups belonging to Firmicutes have the ability to utilize carbohydrates through anerobic fiber fermentation to produce short-chain fatty acids (SCFAs), such as acetates, propionate, and butyrate. These exerts immunomodulatory effects including anti-inflammatory and regulation of gut’s pH to improve the availability of calcium [37–39]. Consistent with their beneficial role in the human gut, we detected an abundance of Firmicutes driven mostly by the genera Clostridium, Faecalibacterium, Blautia and Agathobacter in the gut of the healthy individuals (Fig. 2a, Supplementary Table 2). Additionally, other genera such as Romboutsia, CAG-352, Subdoligranulum, and Dialister (Fig. 2a) were also among the top 20 abundant genera in the healthy individuals. Several bacteria groups belonging to the Ruminococcaceae and Lachnospiraceae families (Fig. 2a) which includes major human butyrate producing gut bacterial species[39] were conspicuously low in TB patients. However, there was an abundance in other Firmicutes of the genera Streptococcus, Enterococcus, Bacteriodes and Erysipelatoclostridium in all the TB patients (Fig. 1a, 1b).
In the TB-DM cohort, the genus Bacteroides was significantly abundant (Fig. 6), comparable to studies which reported a positive association of Bacteroides with diabetic individuals [40–42]. The Bacteroides genera are beneficial for glucose metabolism as well as regulating the expression of tight junction genes in the colon to reduce gut permeability [42,43]. In the TB-Only cohort, the Streptococcus and Erysipelatoclostridium (Fig. 5) which have been associated with high inflammatory conditions [44,45], was abundant while in the TB-HIV in addition to Erysipelatoclostridium, Escherchia-Shigella, Dialister and Corynebacterium were abundant (Fig. 7). Even though most of these bacteria are beneficial in maintaining a healthy gut microbiome, their association with inflammatory conditions could pose some health risk when it is overly abundant in the gut [44].
Contradictory to other studies in Africa which reported reduced abundance in the Actinobacteriota phylum [46–48], interestingly, in this study, we observed an abundance in the Actinobacteriota phylum (9.5%) in the healthy individuals, making it the second most abundant phylum in healthy individuals in this study (Supplementary Fig. 1). The abundance of Actinobacteriota was driven mainly by the Bifidobacterium genera (Fig. 1a, Supplementary table 2) which has been shown to be among the first microbes to colonize the human gut and very beneficial in gut homeostasis and digestion of fiber to produce SCFAs [49]. Contrarily, in all the TB cohorts, Actinobacteria was significantly decreased compared to the controls (Supplementary Fig. 1).
Similar to other studies which associated Prevotella with high fiber-carbohydrate diet mostly in rural areas in sub-Saharan [50,51], we observed an abundance in the genera Prevotella as the dominant group of phylum Bacteriodota in healthy individuals. Among the TB cohorts, Prevotella was more abundant in the TB-DM cohorts (3.4%) (Supplementary table 2) allowing for some association with studies that recorded enrichment of this genera in metabolic disorders such as type 2 diabetes [52,53]. Also, in addition to Prevotella, in the TB-DM cohort, the Bacteriodes which has been reported to be positively associated with diabetes [54,55], was significantly differentially abundant in the TB-DM cohort (Fig. 6).
Irrespective of the overall decrease in microorganism of the Firmicutes, Bacteroidetes and Actinobacteria phyla in all TB cohorts compared to healthy individuals, an enrichment of diverse commensal bacteria of the phylum Proteobacteria containing potential pathogenic bacteria and inflammatory related organisms and other pathobionts was observed. Specifically, the genera Escherichia- Shigella, documented to be abundant in a Ghanaian cohort [51], as well as other African populations [47,56]. Interestingly, in this study, Escherichia- Shigella was not detected as a predominant bacteria in the gut of healthy individuals (Fig. 1a, 1b, Supplementary Fig. 1). This may be attributable to difference in population dynamics including socioeconomic status and dietary habits; the participants in the present study were from urban communities in Accra, the capital city of Ghana, compared to participants from “peri-urban” communities in the previous study [51]. Also, a study in Korea, reported an abundance of the Proteobacteria phylum in type 2 diabetes individuals [57] but no such association was found in this study. We however observed a high abundance of Proteobacteria in TB-HIV individuals (Supplementary table 2) driven mostly by the Escherichia- Shigella family. This genera have been associated with high inflammatory activities as seen in tuberculosis allowing for proliferation of inflammatory-related microorganisms [58] which may not cause disease itself but rather serve to increase susceptibility to intestinal inflammation [45]. Additionally, abundances of Protobacteria have been associated with translocation and inflammation of HIV infected individuals [59].
Intensive phase anti-TB therapy comprises drugs with specific TB targets (HZE) and a broad-spectrum antibiotics (R) [60]. Broad spectrum antibiotics such as RIF, which are active against the majority of bacterial pathogens, could be responsible for the antibiotic associated perturbations [61].
After HRZE treatment, there was higher abundance of bacteria groups in all the follow-up groups, especially in the TB-HIV cohort (Fig. 4b). There was an overall enrichment in the genera Veillonella, Erysipelatoclostridium, and Fusobacterium at follow-up compared to the baseline (Fig. 8), similar to findings from a study reporting abundance of Erysipelatoclostridium in a United States population after HRZE treatment [62]. The genera Erysipelatoclostridium and Fusobacterium are known to be abundant during inflammatory and immune reducing conditions as in TB patients as well as in HIV infection and diabetes [63,64]. While Erysipelatoclostridium possess the ability to flourish during broad-spectrum antibiotic treatment [65], Fusobacterium produces chemical signals that can induce apoptosis in monocytes and macrophages through activation of free fatty acid receptors [66,67], which is also observed in gut mucosa of colorectal cancer patients [68]. Even though the significance of HRZE induced taxonomic perturbations is not well-understood majority of these bacteria have been associated with immune-inducing phenotypes relevant for TB immunity [62].
Contrary, to previous reports which identified vitamin B12 deficiency as a serious problem in diabetics[69–71] due to a negative relationship with metformin, this was not observed in our study population. We found significant abundance of methylmalonyl-CoA mutase in the gut of TB-DM patients compared to controls. This enzyme is involved in Vitamin B12 synthesis, and degradation of amino acids, short-chain fatty acids and cholesterol [72]. Thus, the abundance of this enzyme and MTB’s capacity for de novo biosynthesis of vitamin B12 [73], resulting in reports of elevated B12 vitamin in TB patients [74], could account for our observation. The abundance of methylmalonyl-CoA mutase in only TB-DM group is intriguing and could be the body’s homeostasis activity [75]. Also, a significant abundance of organisms involved in isopropanol biosynthesis in the TB-DM cohort was intriguing, since the metabolite produced is known to inhibit growth of Mycobacterium tuberculosis. However, this abundance was only detected in TB-DM cohort and not in the other TB groups. Additionally, an increase of L-rhamnose mutarotase which is involved in the L-rhamnose metabolism pathway for carbohydrate metabolism [76], and very crucial in the diabetes control [77], was abundant in the TB-DM cohorts.