AUTHOR=Leung Kenneth Siu-Sing , Siu Gilman Kit-Hang , Tam Kingsley King-Gee , To Sabrina Wai-Chi , Rajwani Rahim , Ho Pak-Leung , Wong Samson Sai-Yin , Zhao Wei W. , Ma Oliver Chiu-Kit , Yam Wing-Cheong 

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

JOURNAL=Frontiers in Cellular and Infection Microbiology

VOLUME=7

YEAR=2017

URL=https://www.frontiersin.org/journals/cellular-and-infection-microbiology/articles/10.3389/fcimb.2017.00478

DOI=10.3389/fcimb.2017.00478

ISSN=2235-2988

ABSTRACT=<p><bold>Background:</bold> 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 <italic>in vivo</italic> challenge of anti-TB drugs.</p><p><bold>Result:</bold> 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 <italic>rpoB</italic> S531L, <italic>inhA</italic> C-15T, and <italic>embB</italic> 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 <italic>rv0888</italic> in pre-treatment strain, and a missense mutation at <italic>rv3303c</italic> (<italic>lpdA)</italic> V44I and a 6-bp inframe deletion at codon 67–68 in <italic>rv2071c</italic> (<italic>cobM)</italic> 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.</p><p><bold>Conclusion:</bold> 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 <italic>rv0888, lpdA</italic>, and <italic>cobM</italic> that might explain the difference in antibiotic resistance and growth pattern between the two MDR-TB strains.</p>