Evaluation of single nucleotide polymorphisms in virulence genes of Mycobacterium tuberculosis as markers of lineages and sub-lineages in Tshwane region

Abstract

Tuberculosis (TB) is one of the top ten leading causes of death worldwide with millions of new TB cases reported every year. Understanding the genetic diversity of Mycobacterium tuberculosis (M. tuberculosis) is very crucial for rapid diagnosis and to reduce transmission of TB. Various diagnostic techniques, anti-tuberculosis reagents and vaccination are available, however, the disease is far from being eradicated (Brudey et al., 2006). Mycobacterium tuberculosis is classified into seven major lineages that are key to the most research areas. Recently, multidrug M. tuberculosis have been reported as the most dangerous strains that cause a life-threatening TB. However, the M. tuberculosis with modified virulence and transmissibility, particularly those that are caused by mutations leading to genetic variation and increased pathogenicity are highly reported (Zaychikova et al., 2015). Genetic markers such as variable number tandem repeats, insertion sequence element and direct repeats have been used to

identify lineages. However, the techniques (such as spoligotyping, IS6110-RLFP and MIRU- VNTR) that use these genetic markers have a lot of drawbacks and some have low discriminatory

power (Mikheecheva et al., 2017). Recently, single nucleotide polymorphisms (SNPs) are regarded as the most promising genetic markers for genotyping M. tuberculosis because they have low-level homoplasy and high discriminatory power (Zaychikova et al., 2015). The present study proposed that genotyping M. tuberculosis using polymorphisms in virulence genes may be an alternative approach to determine lineages and may help to detect the M. tuberculosis strains that are epidemiologically dangerous and have adapted to specific geographic regions. This study aimed to identify and evaluate a set of virulence gene SNPs as markers of M. tuberculosis strains circulating in the Tshwane region. A total of 150 susceptible and resistant M. tuberculosis cultures stored in Mycobacteria growth indicator tubes (MGIT) tubes were collected from May to October 2018 at the National Health Laboratory Service, Tshwane Academic Division (NHLS/TAD) to conduct this study. The DNA was extracted using hexadecyltrimethylammonium bromide (CTAB) method and spoligotyping was done to screen for M. tuberculosis lineages. The Beijing and LAM genotypes detected by spoligotyping were sequenced using the Illumina Miseq platform. The bioinformatic analysis of virulence genes in 56 genomes of M. tuberculosis belonging to Beijing and LAM genotypes was performed to detect lineage-specific SNPs markers. Of the 150 M. tuberculosis collected, 57.3% were susceptible M. tuberculosis strains while 42.7% were drug-resistant TB. Spoligotyping of 150 isolates resulted to 86.7% previously shared type (ST) and 13.3% orphans yielding a clustering rate of 63.3%. The Beijing family was found to be the most predominant lineage by 26.7%, followed by T family (16%), LAM (13.3%), East Africa Indian (EAI) (8.7%), S (6%), Manu (4.7%), H (4.7%), CAS (4.0%) and X3 (2.7%). The number of susceptible M. tuberculosis isolates per lineages was higher than drug-resistant TB with isolates detected as Beijing contributing 17.3% of all susceptible isolates, followed by isolates classified as orphans (10%), T family (9.3%), LAM family (8%) and CAS (2.67%). The association between anti-tuberculosis drug-resistant TB and lineages was found in EAI lineage (6.7%), Manu (4%) and S family (3.3%). The family with a high number of isolates which were drug-resistant TB was the EAI1-SOM sub-lineage belonging to the EAI family. 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 (1521 C>T), eccCb1 (1479 G>A), pks5 (6210 G>A), and ponA2 (372 G>T) were identified in the Beijing strains and fadD28 (1392 C>G) within the LAM strains that were not reported in previous studies. Furthermore, this study detected the lineage-specific SNPs: mce3B (145 T>G), eccCb1 (1556 G>T), vapC12 (95 A>G) in Beijing BO/W148 and cyp125 (1076 T>C), mce3B (44 T>C), vapC25 (221 A>C), vapB34 (140 C>A) F15/LAM4/KZN sub-lineages which have been reported to be virulent and associated with drug resistance. This study showed a high genetic diversity of M. tuberculosis strains circulating within the Tshwane region. The Beijing lineage identified in this study was found to be more predominant than the rest of the identified genotypes. This study proposed the alternative method for genotyping M. tuberculosis strains using SNPs in virulence genes of M. tuberculosis. Observations from this study also highlight the advantage of using WGS technique over other genotyping methods such as IS6110-RFLP that has more drawbacks, as most genotypic methods discriminate M. tuberculosis strains using specific genes or regions in the genome of M. tuberculosis while WGS uses the complete genome of M. tuberculosis to determine different M. tuberculosis lineage.