Urinary markers of Mycobacterium tuberculosis and dysbiosis in paediatric tuberculous meningitis cases undergoing treatment

Abstract

Background: The pathogenesis of tuberculous meningitis (TBM) involves infection by Mycobacterium tuberculosis in the meninges and brain. However, recent studies have shown that the immune response and inflammatory processes triggered by TBM can have significant effects on gut microbiota. Disruptions in the gut microbiome have been linked to various systemic consequences, including altered immunity and metabolic dysregulation. Inflammation caused by TBM, antibiotic treatment, and changes in host immunity can all influence the composition of gut microbes. This complex relationship between TBM and the gut microbiome is of great importance in clinical settings. To gain a deeper understanding of the intricate interactions between TBM and the gut microbiome, we report innovative insights into the development of the disease in response to treatment. Ultimately, this could lead to improved outcomes, management strategies and quality of life for individuals affected by TBM.

Method: We used a targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) approach to investigate metabolites associated with gut metabolism in paediatric participants by analysing the urine samples collected from a control group (n = 40), and an experimental group (n = 35) with confirmed TBM, which were subdivided into TBM stage 1 (n = 8), stage 2 (n = 11) and stage 3 (n = 16).

Findings: Our metabolomics investigation showed that, of the 78 initially selected compounds of microbiome origin, eight unique urinary metabolites were identified: 2-methylbutyrlglycine, 3-hydroxypropionic acid, 3-methylcrotonylglycine, 4-hydroxyhippuric acid, 5-hydroxyindoleacetic acid, 5-hydroxyhexanoic acid, isobutyrylglycine, and phenylacetylglutamine as urinary markers of dysbiosis in TBM.

Conclusion: These results - which are supported by previous urinary studies of tuberculosis - highlight the importance of gut metabolism and of identifying corresponding microbial metabolites as novel points for the foundation of improved management of TBM patients.

Keywords: Liquid chromatography-tandem mass spectrometry (LC-MS/MS); Metabolomics; Microbiota; Paediatric; Tuberculous meningitis (TBM); Urine.

Fig. 1

PCA scores plot of initial urinary profile for all 75 metabolites analysed at T1. PCA 1 & 2 explained 48% of the total variation

Fig. 2

Venn diagram of significant metabolites across all six time points of TBM treatment. Number of cases according to TBM stages per time point are given in square brackets. Abbreviations: 2HGA, 2-hydroxyglutaric acid; 3HIA, 3-hydroxyisovaleric acid; 3MGA, 3-methylglutaconic acid; 4HPAA:4-hydroxyphenylacetic acid; AA, aconitic acid; EA, ethylmalonic acid; GA, glucaric acid; HG, hexanoylglycine; ICA, isocitric acid; NAAA, N-acetylaspartic acid; NAP, N-acetylphenylalanine; VA, vanillactic acid; 2HAA, 2-hydroxyadipic acid; 2HPAA, 2-hydroxyphenylacetic acid; 2KGA, 2-ketoglutaric acid; 2MBG, 2-methylbutyrylglycine; 2ODA, 2-octenedioic acid; 2,3PCA, 2,3-pyridinecarboxylic acid; 3HGA, 3-hydroxyglutaric acid; 3HPA, 3-hydroxypropionic acid; 3MCG, 3-methylcrotonylglycine; 3,4DHPAA, 3,4-dihydroxyphenylacetic acid; 4-H-3MMA, 4-hydroxy-3-methoxymandelic acid; 4HPLA, 4-hydroxyphenyllactic acid; 4HHA, 4-hydroxyhippuricacid; 5HIAA, 5-hydroxyindoleacetic acid; 5HHA, 5-hydroxyhexanoic acid; BG, buterylglycine; HA, homovanillic acid; IG, isobutyrylglycine; MCA, methylcitric acid; MSA, methylsuccinic acid; NAT, N acetyltyrosine; PAG, phenylacetylglutamine; PA, pimelic acid; SA, suberic acid; TG, tiglylglycine

Fig. 3

Violin plots of eight significant metabolites at T1: 2-methylbutyrlglycine, 3-hydroxypropionic acid, 3-methylcrotonylglycine, 4-hydroxyhippuric acid, 5-hydroxyindoleacetic acid, 5-hydroxyhexanoic acid, isobutyrylglycine, and phenylacetylglutamine. Control, TBM S1, TBM S2 and TBM S3 consist of 40, 7, 11 and 15 number of samples, respectively. Plots show distribution of data frequency, as well as the median and interquartile ranges. Statistically significant differences (ANOVA Kruskal–Wallis FDR p < 0.01, with multiple corrections) indicated by *

Fig. 4

Linear regression plots with error plots across all six TBM treatment points (T1–T6), for control, TBM_S1, TBM_S2 and TBM_S3, of eight metabolites found to be significant only at T1. All eight metabolites show increased levels at T1, returning to normal (control) levels at final treatment point (T6) for all S2 and S3, while some metabolites remain consistently elevated for S1. Metabolites: 2-methylbutyrlglycine, 3-hydroxypropionic acid, 3-methylcrotonylglycine, 4-hydroxyhippuric acid, 5-hydroxyindoleacetic acid, 5-hydroxyhexanoic acid, isobutyrylglycine, and phenylacetylglutamine

Fig. 5

Branched-chain amino acid (BCAA) catabolism (in the host: red; and as co-metabolism (both host and microbial): purple). Elevated 3-methylcrotonylglycine (3MCG), isobutyrylglycine (IG), and 2-methylbutyrylglycine (2MBG) are urinary markers of dysbiosis at T1 of TBM treatment. It should be noted that the three BCAA intermediates (3-methylbutaonyl-CoA, isobutyrl-CoA and 2-methylbutanoly-CoA) can also be linked to branched-chain fatty acids that are specific to deterioration of the unique cell wall of M. tb (microbial specific: blue). Propionyl-CoA is also an end product that can be linked to Fig. 6

Fig. 6

Microbial-specific (blue background) bi-cycle of acetyl-CoA, propionyl-CoA and carbon fixation via the microbial cycle of hydroxypropionic acid and hydroxybutyric acid. Both these microbial cycles rely upon the intermediary 3HPA, which is elevated and associated with dysbiosis during TBM.