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Title: Comparative genomic analysis of two clonally related multidrug resistant mycobacterium tuberculosis by single molecule real time sequencing
Authors: Leung, KSS
Siu, GKH 
Tam, KKG
Rajwani, R 
Ho, PL
Wong, SSY
Zhao, WW
Yam, WC
Keywords: Multidrug resistance
Mycobacterium tuberculosis
PacBio sequencing
Growth rate
Comparative genomic analysis
Issue Date: 2017
Publisher: Frontiers Research Foundation
Source: Frontiers in cellular and infection microbiology, 15 Nov. 2017, v. 7, 478, p. 1-12 How to cite?
Journal: Frontiers in cellular and infection microbiology 
Abstract: 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.
ISSN: 2235-2988
DOI: 10.3389/fcimb.2017.00478
Rights: Copyright © 2017 Leung, Siu, Tam, To, Rajwani, Ho, Wong, Zhao, Ma and Yam.This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) ( The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
The following publication Leung, K. S. S., Siu, G. K. H., Tam, K. K. G., To, S. W. C., Rajwani, R., Ho, P. L., … & Yam, W. C. (2017). Comparative genomic analysis of two clonally related multidrug resistant mycobacterium tuberculosis by single molecule real time sequencing. Frontiers in Cellular and Infection Microbiology, 7, 478, 1-12 is available at
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