Genetic heterogeneity revealed by sequence analysis of Mycobacterium tuberculosis isolates from extra-pulmonary tuberculosis patients

Abstract

BackgroundTuberculosis remains a major public health problem. Clinical tuberculosis manifests often as pulmonary and occasionally as extra-pulmonary tuberculosis. The emergence of drug resistant tubercle bacilli and its association with HIV is a formidable challenge to curb the spread of tuberculosis. There have been concerted efforts by whole genome sequencing and bioinformatics analysis to identify genomic patterns and to establish a relationship between the genotype of the organism and clinical manifestation of tuberculosis. Extra-pulmonary TB constitutes 15–20 percent of the total clinical cases of tuberculosis reported among immunocompetent patients, whereas among HIV patients the incidence is more than 50 percent. Genomic analysis of M. tuberculosis isolates from extra pulmonary patients has not been explored.ResultsThe genomic DNA of 5 extra-pulmonary clinical isolates of M. tuberculosis derived from cerebrospinal fluid, lymph node fine needle aspirates (FNAC) / biopsies, were sequenced. Next generation sequencing approach (NGS) was employed to identify Single Nucleotide Variations (SNVs) and computational methods used to predict their consequence on functional genes. Analysis of distribution of SNVs led to the finding that there are mixed genotypes in patient isolates and that many SNVs are likely to influence either gene function or their expression. Phylogenetic relationship between the isolates correlated with the origin of the isolates. In addition, insertion sites of IS elements were identified and their distribution revealed a variation in number and position of the element in the 5 extra-pulmonary isolates compared to the reference M. tuberculosis H37Rv strain.ConclusionsThe results suggest that NGS sequencing is able to identify small variations in genomes of M. tuberculosis isolates including changes in IS element insertion sites. Moreover, variations in isolates of M. tuberculosis from non-pulmonary sites were documented. The analysis of our results indicates genomic heterogeneity in the clinical isolates.

High Genotypic Discordance of Concurrent Mycobacterium tuberculosis Isolates from Sputum and Blood of HIV-Infected Individuals

“…Moreover, a recent study showed advanced immune suppression to be associated with increased prevalence of mixed-strain MTB infection[ 36 ] which may lead to selection of the most adapted strain to be disseminated. Although not compared with concurrent pulmonary TB, EPTB has recently been found to be genotypically heterogeneous as revealed by whole genome sequence analysis [ 11 ]. A recent similar study on Mycobacterium avium complex (MAC) found high genetic diversity in strains that cause pulmonary and disseminated disease [ 37 ].…”

Genome-wide Mycobacterium tuberculosis variation (GMTV) database: a new tool for integrating sequence variations and epidemiology

“…Genetic markers that track TB transmission include IS6110, polymorphic GC-reach repetitive sequences, direct repeat regions and mycobacterial interspersed repetitive units [ 3 - 7 ]. Recently, it was shown that bacterial whole genome sequencing (WGS) provides greater discriminative power [ 8 - 11 ]. WGS of multiple isolates may address a broad range of topics – from questions on the transmission of clinical strains to how M. tuberculosis evolves over long and short time scales.…”

A phylogeny-based sampling strategy and power calculator informs genome-wide associations study design for microbial pathogens

“… Zhang et al [ 26 ] 2013 161 Identify drug resistance genes Yes No Phylogenetics and comparison of rates with poisson distribution Yes; list of genes provided Farhat et al [ 15 ] 2013 124 Identify drug resistance genes Yes No Phylogenetics and convergence analysis Yes; list of genes provided Lin et al [ 27 ] 2013 2 Identify drug resistance genes Yes No Comparison with reference mycobacterial strains No Wu et al . [ 28 ] 2013 4 Identify Beijing associated pathways Yes No COG enrichment of genes with snps General pathways rather than individual genes Das et al [ 29 ] 2013 5 Identify genes related to extrapulmonary TB Yes No COG enrichment of genes with snps General pathways rather than individual genes Ilina et al [ 30 ] 2013 4 Identify drug resistance genes Yes No Comparison with reference mycobacterial strains No Abrahams et al [ 31 ] 2013 - Identify resistance targets(s) for novel imidazole No Yes: spontaneous mutants resistant to drug and their sensitive ancestor Identification of all mutations …”