*[1]Danjuma, I., [2]Timan, T. E., 1Daikwo, M. A., and 1Anunobi, O. O.
1Department of Biochemistry, Bingham University, Karu, Nasarawa State, Nigeria
2Zankli Research Center, Bingham University, Karu, Nasarawa State, Nigeria
*Correspondence to: idanjuma4real@gmail.com; +2348063471293
Abstract:
Background: Tuberculosis (TB), caused by Mycobacterium tuberculosis, constitutes a predominant infectious disease with high mortality on a global scale, especially in Nigeria. The emergence of extensively drug-resistant (XDR) TB poses a significant challenge to the control of this disease, underscoring the necessity for the identification of underlying genetic mutations to facilitate informed treatment decisions and mitigate transmission.
Methodology: Sputum samples from a total of 199 patients with validated baseline GeneXpert MTB/rifampicinresistant positive from the northcentral States of Nigeria including Nasarawa, Benue, Kogi, Niger and Abuja in the month of June 2024 were transported to the Zankli TB Reference Laboratory, and processed using NALCNaOH method, followed by centrifugation. The processed samples were inoculated onto prepared Lowenstein Jensen (LJ) media (solid culture) and Mycobacterial Growth Indicator Tube (MGIT) for liquid culture. Ziehl Nelsen (ZN) stain was performed on the isolates of the positive cultures to ascertain the presence of Acid-Fast Bacilli (AFB) and the Bio-line SD AgMPT64 test was performed to confirm the presence of M. tuberculosis. Drug susceptibility testing (DST) was conducted on all positive cultures using the standard proportion method. Long read sequencing was performed on the 2 XDR isolates using the Oxford Nanopore Technology with 50x coverage. The raw reads of the sequenced data were binned into a high-quality metagenome-assembled genome (MAG) via maxbin (V2.2.7), and the quality of the binned metagenome-assembled genome was assessed via checkM (V1.2.3). Resistance genes were discerned through the Comprehensive Antibiotic Resistance Database (CARD) resistance gene identifier, and existing virulence factors were analysed via the VF analyser of the Virulence Factors Database (VFDB), while the phylogenetic tree was constructed via the auto-Multi Locus Species Tree (auto-MLST).
Results: Mutations in katG (R463L, A234V, A431V), rpoB (D516G, H526T, L511R), gyrA (D94N), murA (C117D) and embC (R738Q) were detected in one of the two sequenced XDR isolates (XMTB2) that passed sequencing quality control. The VFDB identified virulence genes such as narG, narH, sapM, and narX, associated with the XMTB2 MAG, while phylogenetic analysis revealed a close phylogenetic connection between the XMTB2 MAG and the well-known Beijing lineage (L2).
Conclusion: The results of this study revealed that the XMTB2 MAG was potentially an XDR isolate that was very virulent because of the presence of many virulence factors identified in it and its close relationship with two of the most widespread and virulent lineages of M. tuberculosis; the Beijing lineage (L2) and the Euro-American lineage (L4).
Keywords: Mycobacterium tuberculosis, XDR TB, mutation, virulence factors, phylogenetic analysis
Download this Article in PDF format below