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

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.

Deciphering Within-Host Microevolution of Mycobacterium tuberculosis through Whole-Genome Sequencing: the Phenotypic Impact and Way Forward

“…Investigating compensatory mechanisms, mutations in genes rpoA and rpoC were shown to appear only after the acquisition of a RIF resistanceconferring mutation in the rpoB gene (47). Another study showed that mutations in the genes Rv0888, Rv2071, and Rv3303c emerged in a highly resistant M. tuberculosis strain during treatment, potentially compensating for an initial decreased fitness of the bacteria (48).…”

“…The missense mutation V44I in lpdA may destabilize the NAD(P)H quinone reductase LpdA (according to the DUET server but not the PROVEAN database) (18,48). This destabilization would change the redox milieu, potentially interfering with the metabolization of the INH prodrug into its active form by the catalase-peroxidase KatG and resulting in an elevation of the INH MIC of the strain (48,54). Trauner et al detected two different lpdA variants present across several isolates from a patient infected with an XDR M. tuberculosis strain, only one of which emerged de novo.…”

“…The SMRT sequencing technology produces long reads that can span these repetitive areas. It uses de novo assembly which allows the detection of rearrangements and copy number variations and the determination of allelic combinations but is prone to high error rates (48,82,83). Future studies should explore these repetitive areas, possibly using a combination of short-read and long-read sequencing to avoid the trade-off between loss of information and increased error rate.…”

MIRU-profiler: a rapid tool for determination of 24-loci MIRU-VNTR profiles from assembled genomes ofMycobacterium tuberculosis

“…In addition, the MIRU-profiler was also tested on four circulating strains from Hong Kong, for which we performed complete genome-sequencing and described in our recent publications ( Leung et al, 2017 ; Rajwani et al, 2018 ). The genomes of these four strains were sequenced on the PacBio RS II platform with average read-length >10 Kb and de-novo assembled into a single contig.…”

Figure 1: Lineage specifi.c sequence variants relative to the reference (H37Rv) gene pks15 (Rv2947c).

“…Studies of Mtb DNA methylation using SMRT sequencing have previously focused on strains originating from Hongkong (2) (Leung et al, 2017) L2, China (12) (Zhu et al, 2015,L2-L4, L6, L8 and more recently a global sample that included Europe, Asia, West Africa and South Africa (16) (Phelan et al, 2018), L1-L2, and L4-L6. In our study the activity of Mtase could be inactivated by four different mutations somewhat in a lineage specific manner.…”