Longitudinal whole genome analysis of pre and post drug treatment Mycobacterium tuberculosis isolates reveals progressive steps to drug resistance

Abstract

Tuberculosis (TB) is one of the leading causes of death due to an infectious disease in the world. Understanding the mechanisms of drug resistance has become pivotal in the detection and treatment of newly emerging resistant TB cases. We have analyzed three pairs of Mycobacterium tuberculosis strains pre- and post-drug treatment to identify mutations involved in the progression of resistance to the drugs rifampicin and isoniazid. In the rifampicin resistant strain, we confirmed a mutation in rpoB (S450L) that is known to confer resistance to rifampicin. We discovered a novel L101R mutation in the katG gene of an isoniazid resistant strain, which may directly contribute to isoniazid resistance due to the proximity of the mutation to the katG isoniazid-activating site. Another isoniazid resistant strain had a rare mutation in the start codon of katG. We also identified a number of mutations in each longitudinal pair, such as toxin-antitoxin mutations that may influence the progression towards resistance or may play a role in compensatory fitness. These findings improve our knowledge of drug resistance progression during therapy and provide a methodology to monitor longitudinal strains using whole genome sequencing, polymorphism comparison, and functional annotation.

Biochemical Characterization of Isoniazid-resistant Mycobacterium tuberculosis: Can the Analysis of Clonal Strains Reveal Novel Targetable Pathways?

“…As it was expected based on previous genomic and western blot analyses [6,12], there was a statistically significant reduction of KatG levels in both clinical and laboratory INHr strains compared to their respective INHs progenitor strains (Table 1, Figure 2). KatG deficiency had been noted in a previous virulence study of these strains in the water-soluble fractions (CYT and CFP) by WB [6].…”

“…It is important to emphasize two points that should be considered in the analysis of the findings presented here, first, with the exception of KatG levels, none of the other protein changes described in this study have associated mutations in their respective genes, as previously revealed by whole genomics sequencing of these strains [12]. Second, this shotgun proteomics approach presents several limitations in that changes in protein abundance may be attributed to decreased expression, differences in protein half-lives, and losses in protein abundance due to aggregation and post-translational modifications with concomitant changes in protein function.…”

“…INHr strains from both clinical and laboratory pairs have the katG mutation V1A, while the clinical INHr strain also bears a second katG mutation, E3V [6,11,12]. A previous whole genome sequencing study on the T lineage corroborated that only katG mutation was associated with drug resistance and the clonality of these pairs [13].…”

“…end of the katG gene [11,12]. These mutations generated an important reduction in KatG levels, which in addition to halting INH activation, also impaired the bacteria's ability to handle oxidative stress, as KatG is an important catalase, peroxidase, and peroxynitritase [47,48].…”

Virulence of Mycobacterium tuberculosis after Acquisition of Isoniazid Resistance: Individual Nature of katG Mutants and the Possible Role of AhpC

“…Mutation analysis in the katG gene previously done by PCR-sequencing [31] confirmed the mutations Val1Ala and Glu3Val for the clinical INHr strain and Val1Ala only for the laboratory INHr strain [30]. The Val1Ala mutation has been previously identified in clinical INHr TB cases [32, 33], while the Glu3Val is a novel mutation.…”