Whole genome sequencing and global metabolome profiling of clinical Mycobacterium tuberculosis isolates provide insights to their drug resistance status
Ashutosh SahooA.K. Das MohapatraHaripriya PriyadarsiniRaghuveer Varma PemmadiAnjan Kumar DasTenzin ChoedonChaitali NikamRajendra Kumar BeheraShyam Kumar MasakapalliRanjan Kumar Nanda
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Abstract Whole genome sequence analysis of the Mycobacterium tuberculosis (Mtb) isolates show correlation to their drug resistance phenotype which may also reflect in their global metabolome. In this report, clinical Mtb isolates (S1, S4, S5, S6, S7, S10) harvested from the sputum of tuberculosis patients were characterized using drug sensitive test (DST), electron microscope, whole genome sequencing (WGS) and metabolomics. Majority of these Mtb isolates showed similar size (length: 1.0–3.2 μm; width: 0.32–0.52 μm) to the H37Rv Mtb strain whereas significant variations were observed in their growth kinetics, WGS and metabolome profiles. In-silico drug resistance prediction, from the WGS data (single-nulceotide polymorphisms (SNP) pattern) of these Mtb isolates showed resistance to tuberculosis drugs and matched with DST results. Differences in the genes involved in stress response, pathogenicity, drug efflux pumps were observed between isolates but genes of the central carbon metabolic pathways and amino acid metabolism were conserved. Gas chromatography and mass spectrometry (GC-MS) based metabolite profiling of these clinical isolates identified 291 metabolites involved in various metabolic pathways and a sub set of these metabolites (glutamic acid, aspartic acid and serine) contributed to the drug resistance patterns. These clinical Mtb isolates could be useful as alternate reagent for understanding host pathogen interaction and the pipeline used for WGS analysis could be used to predict drug resistance pattern of new Mtb isolates.Keywords:
Metabolome
Efflux
Abstract Whole genome sequence analysis of the Mycobacterium tuberculosis (Mtb) isolates show correlation to their drug resistance phenotype which may also reflect in their global metabolome. In this report, clinical Mtb isolates (S1, S4, S5, S6, S7, S10) harvested from the sputum of tuberculosis patients were characterized using drug sensitive test (DST), electron microscope, whole genome sequencing (WGS) and metabolomics. Majority of these Mtb isolates showed similar size (length: 1.0–3.2 μm; width: 0.32–0.52 μm) to the H37Rv Mtb strain whereas significant variations were observed in their growth kinetics, WGS and metabolome profiles. In-silico drug resistance prediction, from the WGS data (single-nulceotide polymorphisms (SNP) pattern) of these Mtb isolates showed resistance to tuberculosis drugs and matched with DST results. Differences in the genes involved in stress response, pathogenicity, drug efflux pumps were observed between isolates but genes of the central carbon metabolic pathways and amino acid metabolism were conserved. Gas chromatography and mass spectrometry (GC-MS) based metabolite profiling of these clinical isolates identified 291 metabolites involved in various metabolic pathways and a sub set of these metabolites (glutamic acid, aspartic acid and serine) contributed to the drug resistance patterns. These clinical Mtb isolates could be useful as alternate reagent for understanding host pathogen interaction and the pipeline used for WGS analysis could be used to predict drug resistance pattern of new Mtb isolates.
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Abstract Much is known regarding the antibiotic susceptibility of planktonic cultures of Mycobacterium tuberculosis, the bacterium responsible for the lung disease tuberculosis (TB). As planktonically-grown M. tuberculosis are unlikely to be entirely representative of the bacterium during infection, we set out to determine how effective a range of anti-mycobacterial treatments were against M. tuberculosis growing as a biofilm, a bacterial phenotype known to be more resistant to antibiotic treatment. Light levels from bioluminescently-labelled M. tuberculosis H37Rv (strain BSG001) were used as a surrogate for bacterial viability, and were monitored before and after 1 week of treatment. After treatment, biofilms were disrupted, washed and inoculated into fresh broth and plated onto solid media to rescue any surviving bacteria. We found that in this phenotypic state M. tuberculosis was resistant to the majority of the compounds tested. Minimum inhibitory concentrations (MICs) increased by 20-fold to greater than 1000-fold, underlying the potential of this phenotype to cause significant problems during treatment.
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Tuberculosis (TB) is a recurrent and progressive disease, with high mortality rates worldwide. The drug-resistance phenomenon of Mycobacterium tuberculosis is a major obstruction of allelopathy treatment. An adverse side effect of allelopathic treatment is that it causes serious health complications. The search for suitable alternatives of conventional regimens is needed, i.e., by considering medicinal plant secondary metabolites to explore anti-TB drugs, targeting the action site of M. tuberculosis. Nowadays, plant-derived secondary metabolites are widely known for their beneficial uses, i.e., as antioxidants, antimicrobial agents, and in the treatment of a wide range of chronic human diseases (e.g., tuberculosis), and are known to "thwart" disease virulence. In this regard, in silico studies can reveal the inhibitory potential of plant-derived secondary metabolites against Mycobacterium at the very early stage of infection. Computational approaches based on different algorithms could play a significant role in screening plant metabolites against disease virulence of tuberculosis for drug designing.
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Tuberculosis remains an important global public health problem, with an estimated prevalence of 14 million individuals with tuberculosis worldwide in 2007. Because antibiotic treatment is one of the main tools for tuberculosis control, knowledge of Mycobacterium tuberculosis drug resistance is an important component for the disease control strategy. Although several gene mutations in specific loci of the M. tuberculosis genome have been reported as the basis for drug resistance, additional resistance mechanisms are now believed to exist. Efflux is a ubiquitous mechanism responsible for intrinsic and acquired drug resistance in prokaryotic and eukaryotic cells. Mycobacterium tuberculosis presents one of the largest numbers of putative drug efflux pumps compared with its genome size. Bioinformatics as well as direct and indirect evidence have established relationships among drug efflux with intrinsic or acquired resistance in M. tuberculosis. This minireview describes the current knowledge on drug efflux in M. tuberculosis.
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Much is known regarding the antibiotic susceptibility of planktonic cultures of Mycobacterium tuberculosis , the bacterium responsible for the lung disease tuberculosis (TB). As planktonically-grown M. tuberculosis are unlikely to be entirely representative of the bacterium during infection, we set out to determine how effective a range of anti-mycobacterial treatments were against M. tuberculosis growing as a biofilm, a bacterial phenotype known to be more resistant to antibiotic treatment. Light levels from bioluminescently-labelled M. tuberculosis H37Rv (strain BSG001) were used as a surrogate for bacterial viability, and were monitored before and after one week of treatment. After treatment, biofilms were disrupted, washed and inoculated into fresh broth and plated onto solid media to rescue any surviving bacteria. We found that in this phenotypic state M. tuberculosis was resistant to the majority of the compounds tested. Minimum inhibitory concentrations (MICs) increased by 20-fold to greater than 1,000-fold, underlying the potential of this phenotype to cause significant problems during treatment.
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The burden and spread of drug-resistant tuberculosis disease is a major public health problem worldwide. The causative agent, Mycobacterium tuberculosis uses several mechanisms to counteract therapy through drugresistance. A major and most common mechanism of drug-resistance is mediated through target mutations. Efflux pumps are emerging as potential agents of drug-resistance and treatment failure. In this review we explore the origin and principles of efflux pump-mediated resistance and determine their impact on second-line drugs used against extensively drug resistant tuberculosis. Inhibition of efflux pumps as a therapeutic intervention is also discussed.
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