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.
