Comparative Genomic Analysis of Two Clonally Related Multidrug Resistant Mycobacterium tuberculosis by Single Molecule Real Time Sequencing

Front Cell Infect Microbiol. 2017 Nov 15:7:478. doi: 10.3389/fcimb.2017.00478. eCollection 2017.


Background: Multidrug-resistant tuberculosis (MDR-TB) is posing a major threat to global TB control. In this study, we focused on two consecutive MDR-TB isolated from the same patient before and after the initiation of anti-TB treatment. To better understand the genomic characteristics of MDR-TB, Single Molecule Real-Time (SMRT) Sequencing and comparative genomic analyses was performed to identify mutations that contributed to the stepwise development of drug resistance and growth fitness in MDR-TB under in vivo challenge of anti-TB drugs. Result: Both pre-treatment and post-treatment strain demonstrated concordant phenotypic and genotypic susceptibility profiles toward rifampicin, pyrazinamide, streptomycin, fluoroquinolones, aminoglycosides, cycloserine, ethionamide, and para-aminosalicylic acid. However, although both strains carried identical missense mutations at rpoB S531L, inhA C-15T, and embB M306V, MYCOTB Sensititre assay showed that the post-treatment strain had 16-, 8-, and 4-fold elevation in the minimum inhibitory concentrations (MICs) toward rifabutin, isoniazid, and ethambutol respectively. The results have indicated the presence of additional resistant-related mutations governing the stepwise development of MDR-TB. Further comparative genomic analyses have identified three additional polymorphisms between the clinical isolates. These include a single nucleotide deletion at nucleotide position 360 of rv0888 in pre-treatment strain, and a missense mutation at rv3303c (lpdA) V44I and a 6-bp inframe deletion at codon 67-68 in rv2071c (cobM) in the post-treatment strain. Multiple sequence alignment showed that these mutations were occurring at highly conserved regions among pathogenic mycobacteria. Using structural-based and sequence-based algorithms, we further predicted that the mutations potentially have deleterious effect on protein function. Conclusion: This is the first study that compared the full genomes of two clonally-related MDR-TB clinical isolates during the course of anti-TB treatment. Our work has demonstrated the robustness of SMRT Sequencing in identifying mutations among MDR-TB clinical isolates. Comparative genome analysis also suggested novel mutations at rv0888, lpdA, and cobM that might explain the difference in antibiotic resistance and growth pattern between the two MDR-TB strains.

Keywords: Mycobacterium tuberculosis; PacBio sequencing; comparative genomic analysis; growth rate; multidrug resistance.

MeSH terms

  • Antitubercular Agents / pharmacology*
  • Antitubercular Agents / therapeutic use
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • Base Sequence
  • Computer Simulation
  • DNA-Directed RNA Polymerases / genetics
  • Extensively Drug-Resistant Tuberculosis / drug therapy
  • Extensively Drug-Resistant Tuberculosis / genetics*
  • Genes, Bacterial / genetics*
  • Genome, Bacterial*
  • Genotype
  • Hong Kong
  • Humans
  • Microbial Sensitivity Tests
  • Mutation
  • Mycobacterium tuberculosis / drug effects*
  • Mycobacterium tuberculosis / genetics*
  • Mycobacterium tuberculosis / growth & development
  • Oxidoreductases / genetics
  • Pentosyltransferases / genetics
  • Phenotype
  • Polymorphism, Genetic
  • Sequence Alignment
  • Sequence Analysis, DNA


  • Antitubercular Agents
  • Bacterial Proteins
  • rpoB protein, Mycobacterium tuberculosis
  • Oxidoreductases
  • InhA protein, Mycobacterium
  • EmbB protein, Mycobacterium tuberculosis
  • Pentosyltransferases
  • DNA-Directed RNA Polymerases