Evaluation of single nucleotide polymorphisms in virulence genes of Mycobacterium tuberculosis as markers of lineages and sub-lineages.
Unarine Matodzia, John Osei. Sekyerea, Nontuthuko E. Maningic, Awelani M. Mutshembeleb
a Department of Medical Microbiology, University of Pretoria, South Africa
b Tuberculosis Platform, South Africa Medical Research Council, South Africa
c Department of Microbiology, School of Life Science, College of Agriculture, Engineering, and Science, University of Kwazulu-Natal Westville Campus, South Africa

Background: Understanding genetic diversity of Mycobacterium tuberculosis (M. tuberculosis) is very crucial for rapid diagnosis and to reduce transmission of tuberculosis. This study determines lineage specific SNPs within M. tuberculosis virulence genes and lineage distribution amongst M. tuberculosis obtained in the Tshwane region.
Methods: One hundred and fifty M. tuberculosis isolates were collected and sub-cultured on MGIT 960 machine. DNA was extracted using CTAB method and spoligotyping was done to screen for M. tuberculosis lineages. Beijing and LAM lineages which were detected by Spoligotyping were further genotyped by whole-genome sequencing (WGS) using Illumina Miseq platform.
Results: Of the 150 isolates 86.7% were previously shared type and 13.3% were orphans yielding a clustering rate of 63.3%. The Beijing family was found to be the most predominant by 26.7%, followed by T family (16%), LAM (13.3%), EAI (8.7%), S (6%), Manu (4.7%), H (4.7%), CAS (4.0%) and X3 (2.7%). This study successfully identified 29 Beijing and 6 LAM signature SNPs that can be used to classify clinical M. tuberculosis isolates. Within these signature SNPs, fadD28 (C1521T), eccCb1 (G1479A), pks5 (G6210A), and ponA2 (G372T) were identified in the Beijing strains and fadD28 (C1392G) within the LAM strains that were not reported in previous studies. Furthermore, this study detected the lineage-specific SNPs: mce3B (T145G), eccCb1 (G1556T), vapC12 (A95G) in Beijing BO/W148 and cyp125 (T1076 C), mce3B (T44C), vapC25 (A221C), vapB34 (C140A) F15/LAM4/KZN sub-lineages which have been reported to be virulent and associated with drug resistance.
Conclusion: This study proposed the alternative method for genotyping M. tuberculosis strains using SNPs in virulence genes. This study also highlight the advantage of using WGS technique over other genotyping methods such as IS6110-RFLP that have more drawbacks, as most methods detect the M. tuberculosis strains using either a specific genes or regions in the genome to determine different lineage.

Unarine Matodzi MSc

University of Pretoria

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