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.
Highlights
Tuberculosis (TB) caused by Mycobacterium tuberculosis (MTB) has been a global public health challenge for decades
MYCOTB Sensititre assay showed that the Minimum inhibitory concentrations (MICs) for pretreatment and post-treatment strains were within ±1 doubling dilution in RIF, STR, MOX, OFX, AMI, KAN, ETH, para-aminosalicylic acid (PAS), and CYS which was concordant to the initial phenotypic drug susceptibility tests (DSTs) result from sputum culture
MGIT PZA kit showed that both MDRTB strains were resistant to PZA at 100 mg/L the post-treatment strain showed 4-fold elevation in EMB MIC (8– 32 mg/L), 8-fold elevation in INH MIC (0.5–4 mg/L), and 16fold elevation in RFB MIC (1–16 mg/L) when compared to the pre-treatment strain (Table 2)
Summary
Tuberculosis (TB) caused by Mycobacterium tuberculosis (MTB) has been a global public health challenge for decades. Despite the enormous global efforts in TB control, multidrug resistant TB (MDR-TB) with resistance to at least rifampicin (RIF) and isoniazid (INH) is starting to threaten the treatment regimens currently available. In 2016, World Health Organization estimated a total of 480,000 TB cases caused by MDR-TB. Among these MDR-TB cases, ∼9.5% were extensively drug resistant TB (XDR-TB) with additional resistance toward fluoroquinolones and one of the other secondline injectable anti-TB drugs. 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
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