Cascade of deoxyribozymes for the colorimetric analysis of drug resistance in Mycobacterium tuberculosis.
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ABSTRACT:
SUBMITTER: Dhar BC
PROVIDER: S-EPMC7484165 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundEmergence of drug resistant varieties of tuberculosis is posing a major threat to global tuberculosis eradication programmes. Although several approaches have been explored to counter resistance, there has been limited success due to a lack of understanding of how resistance emerges in bacteria upon drug treatment. A systems level analysis of the proteins involved is essential to gain insights into the routes required for emergence of drug resistance.ResultsWe derive a genome-scale protein-protein interaction network for Mycobacterium tuberculosis H37Rv from the STRING database, with proteins as nodes and interactions as edges. A set of proteins involved in both intrinsic and extrinsic drug resistance mechanisms are identified from literature. We then compute shortest paths from different drug targets to the set of resistance proteins in the protein-protein interactome, to derive a sub-network relevant to study emergence of drug resistance. The shortest paths are then scored and ranked based on a new scheme that considers (a) drug-induced gene upregulation data, from microarray experiments reported in literature, for the individual nodes and (b) edge-hubness, a network parameter which signifies centrality of a given edge in the network. High-scoring paths identified from this analysis indicate most plausible pathways for the emergence of drug resistance. Different targets appear to have different propensities for four drug resistance mechanisms. A new concept of 'co-targets' has been proposed to counter drug resistance, co-targets being defined as protein(s) that need to be simultaneously inhibited along with the intended target(s), to check emergence of resistance to a given drug.ConclusionThe study leads to the identification of possible pathways for drug resistance, providing novel insights into the problem of resistance. Knowledge of important proteins in such pathways enables identification of appropriate 'co-targets', best examples being RecA, Rv0823c, Rv0892 and DnaE1, for drugs targeting the mycolic acid pathway. Insights obtained about the propensity of a drug to trigger resistance will be useful both for more careful identification of drug targets as well as to identify target-co-target pairs, both implementable in early stages of drug discovery itself. This approach is also inherently generic, likely to significantly impact drug discovery.
| S-EPMC2649132 | biostudies-literature
Project description:Mycobacterium tuberculosis adapts to the environment by selecting for advantageous single-nucleotide polymorphisms (SNPs). We studied whether advantageous SNPs could be distinguished from neutral mutations within genes associated with drug resistance. A total of 1,003 clinical isolates of M. tuberculosis were related phylogenetically and tested for the distribution of SNPs in putative drug resistance genes. Drug resistance-associated versus non-drug-resistance-associated SNPs in putative drug resistance genes were compared for associations with single versus multiple-branch outcomes using the chi-square and Fisher exact tests. All 286 (100%) isolates containing isoniazid (INH) resistance-associated SNPs had multibranch distributions, suggestive of multiple ancestry and convergent evolution. In contrast, all 327 (100%) isolates containing non-drug-resistance-associated SNPs were monophyletic and thus showed no evidence of convergent evolution (P < 0.001). Convergence testing was then applied to SNPs at position 481 of the iniA (Rv0342) gene and position 306 of the embB gene, both potential drug resistance targets for INH and/or ethambutol. Mutant embB306 alleles showed multibranch distributions, suggestive of convergent evolution; however, all 44 iniA(H481Q) mutations were monophyletic. In conclusion, this study validates convergence analysis as a tool for identifying mutations that cause INH resistance and explores mutations in other genes. Our results suggest that embB306 mutations are likely to confer drug resistance, while iniA(H481Q) mutations are not. This approach may be applied on a genome-wide scale to identify SNPs that impact antibiotic resistance and other types of biological fitness.
| S-EPMC2533473 | biostudies-literature
Project description:BackgroundContinuing evolution of the Mycobacterium tuberculosis (Mtb) complex genomes associated with resistance to anti-tuberculosis drugs is threatening tuberculosis disease control efforts. Both multi- and extensively drug resistant Mtb (MDR and XDR, respectively) are increasing in prevalence, but the full set of Mtb genes involved are not known. There is a need for increased sensitivity of genome-wide approaches in order to elucidate the genetic basis of anti-microbial drug resistance and gain a more detailed understanding of Mtb genome evolution in a context of widespread antimicrobial therapy. Population structure within the Mtb complex, due to clonal expansion, lack of lateral gene transfer and low levels of recombination between lineages, may be reducing statistical power to detect drug resistance associated variants.ResultsTo investigate the effect of lineage-specific effects on the identification of drug resistance associations, we applied genome-wide association study (GWAS) and convergence-based (PhyC) methods to multiple drug resistance phenotypes of a global dataset of Mtb lineages 2 and 4, using both lineage-wise and combined approaches. We identify both well-established drug resistance variants and novel associations; uniquely identifying associations for both lineage-specific and -combined GWAS analyses. We report 17 potential novel associations between antimicrobial resistance phenotypes and Mtb genomic variants.ConclusionsFor GWAS, both lineage-specific and -combined analyses are useful, whereas PhyC may perform better in contexts of greater diversity. Unique associations with XDR in lineage-specific analyses provide evidence of diverging evolutionary trajectories between lineages 2 and 4 in response to antimicrobial drug therapy.
| S-EPMC6440112 | biostudies-literature
Project description:As the causative bacteria of tuberculosis, Mycobacteriumtuberculosis (M. tb) is aggravated by the emergence of its multidrug-resistant isolates in China. Mutations of six of the most frequently reported resistant genes (rpoB, katG, inhA, embB, gyrA, and rpsL) were detected for rifampicin (RIF), isoniazid (INH), ethambutol (EMB), ofloxacin (OFX), and streptomycin (STR) in this study. The amino acid missense mutations (MMs) and their corresponding single nucleotide polymorphism mutations for all drug-resistant (DR) isolates are described in detail. All isolates were divided into non-extensively drug-resistant (Non-XDR) and preXDR/XDR groups. No statistical differences were detected among MMs and linked MMs (LMs) between the two groups, except for rpsL 88 (p = 0.037). In the preXDR/XDR group, the occurrence of MMs in rpoB, katG, and inhA developed phenotypic resistance and MMs of rpoB 531, katG 315, rpsL 43, and rpsL 88 could develop high levels of DR. It is necessary to carry out epidemiological investigations of DR gene mutations in the local region, and thus provide necessary data to support the design of new technologies for rapid detection of resistant M. tb and the optimization of detection targets.
| S-EPMC8614678 | biostudies-literature
Project description:Although pyrazinamide (PZA) is a key component of first- and second-line tuberculosis treatment regimens, there is no gold standard to determine PZA resistance. Approximately 50% of multidrug-resistant tuberculosis (MDR-TB) and over 90% of extensively drug-resistant tuberculosis (XDR-TB) strains are also PZA resistant. pncA sequencing is the endorsed test to evaluate PZA susceptibility. However, molecular methods have limitations for their wide application. In this study, we standardized and evaluated a new method, MODS-Wayne, to determine PZA resistance. MODS-Wayne is based on the detection of pyrazinoic acid, the hydrolysis product of PZA, directly in the supernatant of sputum cultures by detecting a color change following the addition of 10% ferrous ammonium sulfate. Using a PZA concentration of 800?µg/ml, sensitivity and specificity were evaluated at three different periods of incubation (reading 1, reading 2, and reading 3) using a composite reference standard (MGIT-PZA, pncA sequencing, and the classic Wayne test). MODS-Wayne was able to detect PZA resistance, with a sensitivity and specificity of 92.7% and 99.3%, respectively, at reading 3. MODS-Wayne had an agreement of 93.8% and a kappa index of 0.79 compared to the classic Wayne test, an agreement of 95.3% and kappa index of 0.86 compared to MGIT-PZA, and an agreement of 96.9% and kappa index of 0.90 compared to pncA sequencing. In conclusion, MODS-Wayne is a simple, fast, accurate, and inexpensive approach to detect PZA resistance, making this an attractive assay especially for low-resource countries, where TB is a major public health problem.
| S-EPMC6355525 | biostudies-literature
Project description:Tuberculosis (TB) is the leading cause of death by an infectious disease, and global TB control efforts are increasingly threatened by drug resistance in Mycobacterium tuberculosis. Unlike most bacteria, where lateral gene transfer is an important mechanism of resistance acquisition, resistant M. tuberculosis arises solely by de novo chromosomal mutation. Using whole-genome sequencing data from two natural populations of M. tuberculosis, we characterized the population genetics of known drug resistance loci using measures of diversity, population differentiation, and convergent evolution. We found resistant subpopulations to be less diverse than susceptible subpopulations, consistent with ongoing transmission of resistant M. tuberculosis. A subset of resistance genes ("sloppy targets") were characterized by high diversity and multiple rare variants; we posit that a large genetic target for resistance and relaxation of purifying selection contribute to high diversity at these loci. For "tight targets" of selection, the path to resistance appeared narrower, evidenced by single favored mutations that arose numerous times in the phylogeny and segregated at markedly different frequencies in resistant and susceptible subpopulations. These results suggest that diverse genetic architectures underlie drug resistance in M. tuberculosis and that combined approaches are needed to identify causal mutations. Extrapolating from patterns observed for well-characterized genes, we identified novel candidate variants involved in resistance. The approach outlined here can be extended to identify resistance variants for new drugs, to investigate the genetic architecture of resistance, and when phenotypic data are available, to find candidate genetic loci underlying other positively selected traits in clonal bacteria. IMPORTANCEMycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a significant burden on global health. Antibiotic treatment imposes strong selective pressure on M. tuberculosis populations. Identifying the mutations that cause drug resistance in M. tuberculosis is important for guiding TB treatment and halting the spread of drug resistance. Whole-genome sequencing (WGS) of M. tuberculosis isolates can be used to identify novel mutations mediating drug resistance and to predict resistance patterns faster than traditional methods of drug susceptibility testing. We have used WGS from natural populations of drug-resistant M. tuberculosis to characterize effects of selection for advantageous mutations on patterns of diversity at genes involved in drug resistance. The methods developed here can be used to identify novel advantageous mutations, including new resistance loci, in M. tuberculosis and other clonal pathogens.
| S-EPMC5790871 | biostudies-literature
Project description:The emergence of antibiotic resistance in Mycobacterium tuberculosis has raised the concern that pathogen strains that are virtually untreatable may become widespread. The acquisition of resistance to antibiotics results in a longer duration of infection in a host, but this resistance may come at a cost through a decreased transmission rate. This raises the question of whether the overall fitness of drug-resistant strains is higher than that of sensitive strains--essential information for predicting the spread of the disease. Here, we directly estimate the transmission cost of drug resistance, the rate at which resistance evolves, and the relative fitness of resistant strains. These estimates are made by using explicit models of the transmission and evolution of sensitive and resistant strains of M. tuberculosis, using approximate Bayesian computation, and molecular epidemiology data from Cuba, Estonia, and Venezuela. We find that the transmission cost of drug resistance relative to sensitivity can be as low as 10%, that resistance evolves at rates of approximately 0.0025-0.02 per case per year, and that the overall fitness of resistant strains is comparable with that of sensitive strains. Furthermore, the contribution of transmission to the spread of drug resistance is very high compared with acquired resistance due to treatment failure (up to 99%). Estimating such parameters directly from in vivo data will be critical to understanding and responding to antibiotic resistance. For instance, projections using our estimates suggest that the prevalence of tuberculosis may decline with successful treatment, but the proportion of cases associated with resistance is likely to increase.
| S-EPMC2732896 | biostudies-literature
Project description:Rapid and accurate detection of tuberculosis (TB) drug resistance is critical for the successful treatment and control of TB. Here, we investigated resistance to anti-TB drugs and genetic variations in 215 drug-resistant Mycobacterium tuberculosis isolates in Korea. Genetic variations were observed in rpoB Ser531Leu, katG Ser315Thr, and gyrA Asp94Gly; however, the minimum inhibitory concentrations varied, which can be attributed to other resistance mechanisms. Examination of genetic relatedness among drug-resistant isolates revealed that the cluster size of resistant bacteria was less than six strains, suggesting no evidence of a large-scale epidemic caused by a specific strain. However, rpoC mutants of the rifampicin-resistant isolates were composed of five types of clusters, suggesting that these compensatory mutations advance propagation. In the present study, more than 90% of the resistance mechanisms to major anti-TB drugs were identified, and the effect of each mutation on drug resistance was estimated. With the clinical application of recent next-generation sequencing-based susceptibility testing, the present study is expected to improve the clinical utilization of genotype-based drug susceptibility testing for the diagnosis and treatment of patients with drug-resistant TB.
| S-EPMC10451913 | biostudies-literature
Project description:Persistence, manifested as drug tolerance, represents a significant obstacle to global tuberculosis control. The bactericidal drugs isoniazid and rifampicin kill greater than 99% of exponentially growing Mycobacterium tuberculosis (Mtb) cells, but the remaining cells are persisters, cells with decreased metabolic rate, refractory to killing by these drugs, and able to generate drug-resistant mutants. We discovered that the combination of cysteine or other small thiols with either isoniazid or rifampicin prevents the formation of drug-tolerant and drug-resistant cells in Mtb cultures. This effect was concentration- and time-dependent, relying on increased oxygen consumption that triggered enhanced production of reactive oxygen species. In infected murine macrophages, the addition of N-acetylcysteine to isoniazid treatment potentiated the killing of Mtb Furthermore, we demonstrate that the addition of small thiols to Mtb drug treatment shifted the menaquinol/menaquinone balance toward a reduced state that stimulates Mtb respiration and converts persister cells to metabolically active cells. This prevention of both persister cell formation and drug resistance leads ultimately to mycobacterial cell death. Strategies to enhance respiration and initiate oxidative damage should improve tuberculosis chemotherapies.
| S-EPMC5410800 | biostudies-literature
Project description:Tuberculosis is a chronic infectious disease caused by Mycobacterium tuberculosis (Mtb). Due to the extensive use of anti-tuberculosis drugs and the development of mutations, the emergence and spread of multidrug-resistant tuberculosis is recognized as one of the most dangerous threats to global tuberculosis control. Some single mutations have been identified to be significantly linked with drug resistance. However, the prior research did not take gene-gene interactions into account, and the emergence of transmissible drug resistance is connected with multiple genetic mutations. In this study we use the bioinformatics software GBOOST (The Hong Kong University, Clear Water Bay, Kowloon, Hong Kong, China) to calculate the interactions of Single Nucleotide Polymorphism (SNP) pairs and identify gene pairs associated with drug resistance. A large part of the non-synonymous mutations in the drug target genes that were included in the screened gene pairs were confirmed by previous reports, which lent sound solid credits to the effectiveness of our method. Notably, most of the identified gene pairs containing drug targets also comprise Pro-Pro-Glu (PPE) family proteins, suggesting that PPE family proteins play important roles in the drug resistance of Mtb. Therefore, this study provides deeper insights into the mechanisms underlying anti-tuberculosis drug resistance, and the present method is useful for exploring the drug resistance mechanisms for other microorganisms.
| S-EPMC5037696 | biostudies-literature