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

Frontiers in Cellular and Infection Microbiology

Rajwani, Rahim¹  Siu, Gilman Kit-Hang¹  Yam, Wing-Cheong²  Tam, Kingsley King-Gee²  Ho, Pak-Leung²  Leung, Kenneth Siu-Sing²  Wong, Samson Sai-Yin²  To, Sabrina Wai-Chi²  Zhao, Wei W.³  Ma, Oliver Chiu-Kit³ 

[1] Department of Health Technology and Informatics, The Hong Kong Polytechnic University, Hong Kong;Department of Microbiology, Queen Mary Hospital, The University of Hong Kong, Hong Kong;KingMed Diagnostics, Science Park, Hong Kong

关键词: multidrug resistance;    Mycobacterium tuberculosis;    PacBio Sequencing;    growth rates;    Comparative genomic analysis;   

DOI  :  10.3389/fcimb.2017.00478

学科分类：生物科学（综合）

来源: Frontiers

【 摘 要 】

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 towards 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-, eight- and four-fold elevation in the minimum inhibitory concentrations (MICs) towards 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.

【 授权许可】

CC BY