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Whole Genome Sequencing of Mycobacterium tuberculosis Clinical Isolates From India Reveals Genetic Heterogeneity and Region-Specific Variations That Might Affect Drug Susceptibility

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Whole Genome Sequencing of Mycobacterium tuberculosis Clinical Isolates From India Reveals Genetic Heterogeneity and Region-Specific Variations That Might Affect Drug Susceptibility

Jayshree Advani et al. Front Microbiol.

Abstract

Whole genome sequencing (WGS) of Mycobacterium tuberculosis has been constructive in understanding its evolution, genetic diversity and the mechanisms involved in drug resistance. A large number of sequencing efforts from across the globe have revealed genetic diversity among clinical isolates and the genetic determinants for their resistance to anti-tubercular drugs. Considering the high TB burden in India, the availability of WGS studies is limited. Here we present, WGS results of 200 clinical isolates of M. tuberculosis from North India which are categorized as sensitive to first-line drugs, mono-resistant, multi-drug resistant and pre-extensively drug resistant isolates. WGS revealed that 20% of the isolates were co-infected with M. tuberculosis and non-tuberculous mycobacteria species. We identified 12,802 novel genetic variations in M. tuberculosis isolates including 343 novel SNVs in 38 genes which are known to be associated with drug resistance and are not currently used in the diagnostic kits for detection of drug resistant TB. We also identified M. tuberculosis lineage 3 to be predominant in the northern region of India. Additionally, several novel SNVs, which may potentially confer drug resistance were found to be enriched in the drug resistant isolates sampled. This study highlights the significance of employing WGS in diagnosis and for monitoring further development of MDR-TB strains.

Keywords: fluoroquinolones; metagenomics; molecular genotyping; mycobacterial genetic heterogeneity; next generation sequencing.

Figures

FIGURE 1
FIGURE 1
Isolates identified as co-infections. The length of bars indicates proportion of reads that were aligned to corresponding mycobacterial species. (WHO definitions for case history. (i) New case- Patient has never been treated for TB. (ii) Retreatment case- Patient on retreatment regimen with first-line drugs. (iii) Second-line treatment- Patient has been started directly on second-line treatment for MDR-TB or RR-TB, without being started on a first line drugs. (iv) Relapse case- Patient has been previously treated for TB and are now diagnosed with a recurrent episode of TB. (v) Not known- Case history unavailable).
FIGURE 2
FIGURE 2
Genetic variations in Mycobacterium tuberculosis isolates. (A) Circos plot depicting novel SNVs and INDELS. (B) Principal Component Analysis (PCA) of non-synonymous SNVs from GMTV, isolates from South India and isolates from the current study (North India). Blue, green, and pink dots represent isolates from North India, South India and GMTV database, respectively.
FIGURE 3
FIGURE 3
Distribution of isolates. (A) Phylogenetic clustering of M. tuberculosis isolates from North and South India. (B) Geographical distribution of different M. tuberculosis lineages across the globe.
FIGURE 4
FIGURE 4
Frequency of SNVs that confer drug resistance. (A) Frequency of SNVs known to confer resistance against first and second-line anti-tubercular drugs in clinical isolates from North India. (B) Frequency of novel SNVs in genes that confer drug resistance.
FIGURE 5
FIGURE 5
Single nucleotide variations in genes that confer resistance to fluoroquinolone and bedaquiline. (A) SNVs in quinolone resistance-determining regions (QRDRs) of gyrA and gyrB genes. SNVs marked in blue are reported in isolates obtained from South India and SNVs marked in red are the ones identified in our study. (B) SNVs in Rv0678 gene that potentially confer bedaquiline resistance. SNVs marked in blue are reported earlier and SNVs marked in red are novel.
FIGURE 6
FIGURE 6
Number of SNVs in rpo A, B, and C genes in different lineages in isolates from North and South India. (A) Colored bands represent number of SNVs in rpo A, B, and C genes in isolates from North India across different lineages. (B) Colored bands represent number of SNVs in rpo A, B, and C genes in isolates from South India across different lineages.

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