Drug-resistant Mycobacterium Tuberculosis Research Articles

Mycobacterium tuberculosis sRNA MTS2823 regulates the growth of the multidrug-resistant strain in macrophages.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (MTB), is a serious contagious disease. MTB-encoded small regulatory RNA (sRNA) MTS2823 was reported to be upregulated in the plasma of TB patients. Nevertheless, whether MTS2823 is implicated in MTB drug resistance is unclear. Human macrophage cell line THP-1 was infected with the drug-susceptible strain H37Rv or the multidrug-resistant (MDR) strain 8462. Colony-forming unit assay was implemented for evaluating intracellular growth of the MTB strains. Enzyme-linked immunosorbent assay was used for measurement of inflammatory cytokines. Real-time quantitative polymerase chain reaction was utilized to assess MTS2823 and recombinase A (recA) expression in strains 8462 and H37Rv. Nitric oxide (NO) production in the MDR strain-infected THP-1 cells was measured. In this study, MTS2823 was found to display a low level in the MDR strain. Overexpressing MTS2823 promoted intracellular growth of the MDR strain and inhibited inflammatory cytokine and NO production in infected THP-1 cells. RecA might be a target of MTS2823 in the MDR strain. Overall, MTB-encoded sRNA MTS2823 displays a low level and regulates the growth of the MDR strain in THP-1 cells by modulating recA.

Immune Disorders in Patients with Pulmonary Tuberculosis with Primary and Acquired Drug-Resistance of <i>Mycobacterium Tuberculosis</i>

The objective: to evaluate main parameters of cellular, humoral immunity and natural resistance in tuberculosis patients with primary and acquired multiple drug resistance; to study the correlation of these parameters with clinical and radiological manifestations.Subjects and Methods. A prospective study was conducted which included 169 patients with pulmonary tuberculosis, 80 of them were new cases of drug susceptible pulmonary tuberculosis and they made the Comparison Group (CG), and 89 patients suffered from multiple drug resistant tuberculosis and they made the Main Group (MG). The Main Group was divided into 2 subgroups: MG-1 – 40 patients with primary multiple drug resistant tuberculosis; MG-2 ‒ 49 patients who acquired drug resistance of Mycobacterium tuberculosis during treatment of tuberculosis.The immune assays included the lymphocyte blast transformation reaction (LBTR) to phytohemagglutinin and tuberculin (PPD), immunophenotyping of CD3+ and CD19+ cells; phagocytic index and phagocytic number were calculated; nitro blue tetrazolium test was used, concentrations of immunoglobulins and antimycobacterial antibodies were determined.Results. The clinical laboratory data demonstrated a more severe course of the disease in patients with multiple drug resistant tuberculosis, especially in those with acquired multiple drug resistant tuberculosis which was confirmed by the severity of immune deficiencies of the cellular immunity and innate resistance as well as by the overactivity of humoral immunity. The levels of CD3+ and LBTR reaction to PPD had a negative correlation with clinical manifestations and radiological features demonstrating the severity of pulmonary tuberculosis in patients with multiple drug resistance, especially in those with acquired multiple drug resistance; and CD19+ and antimycobacterial antibodies positively correlated with clinical manifestations and radiological signs.Conclusions. Parameters of cellular immunity and natural resistance were the lowest in the patients with acquired multiple drug resistance of Mycobacterium tuberculosis versus primary multiple drug resistance and drug susceptible Mycobacterium tuberculosis. Parameters of humoral immunity demonstrated the increased activity to compensate for the insufficiency of cellular immunity regardless of the type of resistance of Mycobacterium tuberculosis.

Analysis of molecular mechanisms of drug resistance of Mycobacterium tuberculosis in patients with pulmonary tuberculosis and its pharmacoeconomics

Purpose: To investigate the molecular mechanisms of drug resistance of Mycobacterium tuberculosis in patients with pulmonary tuberculosis and its pharmacoeconomics. 
 Methods: Data pertaining to patients with primary tuberculosis treated in the First Affiliated Hospital of Zhaoqing Medical College, Zhaoqing, China from January 2020 to June 2021 were retrospectively analyzed. Sputum specimens were collected from all eligible patients, and 151 uncontaminated specimens with good bacteriophage activity were screened. 
 Results: A total of 107 Mycobacterium tuberculosis strains were isolated from the 151 specimens, 31 of which strains were resistant to varying degrees to rifampicin, isoniazid, streptomycin, and ethambutol with an overall resistance of 28.97 %. There were 16 strains with rpoB mutation, 22 strains with katG mutation, and 8 strains with inhA mutation. The difference in the sputum negative rate, lesion absorption rate, and tuberculosis cavity closure rate, and total medical cost between the two group were not statistically significant (p > 0.05). The incidence of adverse reactions in the FDC group was significantly lower than that in the blister pack group (p < 0.05). 
 Conclusion: The total resistance of Mycobacterium tuberculosis in primary tuberculosis patients remains at a high level, and the development of resistance is associated with mutations in rpoB, katG, and inhA genes. FDC regimen provides more pharmacoeconomic and therapeutic benefits than blister pack regimen.

MmpL3 Inhibition as a Promising Approach to Develop Novel Therapies against Tuberculosis: A Spotlight on SQ109, Clinical Studies, and Patents Literature.

Tuberculosis (TB) is accountable for considerable global morbidity and mortality. Effective TB therapy with multiple drugs completes in about six months. The longer duration of TB therapy challenges patient compliance and contributes to treatment collapse and drug resistance (DR) progress. Therefore, new medications with an innovative mechanism of action are desperately required to shorten the TB therapy's duration and effective TB control. The mycobacterial membrane protein Large 3 (MmpL3) is a novel, mycobacteria-conserved and recognized promiscuous drug target used in the development of better treatments for multi-drug resistance TB (MDR-TB) and extensively drug-resistant TB (XDR-TB). This article spotlights MmpL3, the clinical studies of its inhibitor (SQ109), and the patent literature. The literature on MmpL3 inhibitors was searched on PubMed and freely available patent databases (Espacenet, USPTO, and PatentScope). SQ109, an analog of ethambutol (EMB), is an established MmpL3 inhibitor and has completed Phase 2b-3 clinical trials. Infectex and Sequella are developing orally active SQ109 in partnership to treat MDR pulmonary TB. SQ109 has demonstrated activity against drug-sensitive (DS) and drug-resistant (DR) Mycobacterium tuberculosis (Mtb) and a synergistic effect with isoniazid (INH), rifampicin (RIF), clofazimine (CFZ), and bedaquiline (BNQ). The combination of SQ109, clofazimine, bedaquiline, and pyrazinamide (PZA) has been patented due to its excellent anti-TB activity against MDR-TB, XDR-TB, and latent-TB. The combinations of SQ109 with other anti-TB drugs (chloroquine, hydroxychloroquine, and sutezolid) have also been claimed in the patent literature. SQ109 is more potent than EMB and could substitute EMB in the intensive stage of TB treatment with the three- or four-drug combination. Developing MmpL3 inhibitors is a promising approach to fighting the challenges associated with DS-TB and DR-TB. The authors foresee MmpL3 inhibitors such as SQ109 as future drugs for TB treatment.

Characterizing in vivo loss of virulence of an HN878 Mycobacterium tuberculosis isolate from a genetic duplication event

The increase of global cases of drug resistant (DR) Mycobacterium tuberculosis (M.tb) is a serious problem for the tuberculosis research community and the goals to END TB by 2030. Due to the need for advancing and screening next generation therapeutics and vaccines, we aimed to design preclinical DR models of Beijing lineage M.tb HN878 strain in different mouse backgrounds. We found escalating sensitivities of morbidity due to low dose aerosol challenge (50–100 bacilli) in CB6F1, C57BL/6 and SWR mice, respectively. We also observed that pulmonary bacterial burden at morbidity endpoints correlated inversely with survival over time between mouse strains. Interestingly, with in vitro passaging and in the process of selecting individual DR mutant colonies, we observed a significant decrease in in vivo HN878 strain virulence, which correlated with the acquisition of a large genetic duplication. We confirmed that low passage infection stocks with no or low prevalence of the duplication, including stocks directly acquired from the BEI resources biorepository, retained virulence, measured by morbidity over time. These data help confirm previous reports and emphasize the importance of monitoring virulence and stock fidelity.

Strategies Employed to Evade the Host Immune Response and the Mechanism of Drug Resistance in Mycobacterium tuberculosis: In Search of Finding New Targets.

The partial effectiveness of the host immune response to M. tuberculosis drives bacteria into a latent state, but it is difficult to eliminate the bacteria completely. Usually, this latent condition of M. tuberculosis is reversible, and reactivation of tuberculosis is the leading cause of the majority of transmission. A number of studies performed on animal models and humans have not yet provided a detailed understanding of the mechanisms or correlates of immunity of M. tuberculosis infection or why there is a significant immunity failure to remove the pathogen. Moreover, the mechanism of resistance involved in drug-resistant M. tuberculosis leads to the emergence of strains of bacteria that show significant resistance to the majority of anti-tuberculosis drugs. We have also provided the recent findings and trends regarding the development of new drug molecules to treat drug and multidrug-resistant tuberculosis and the advancements in immunotherapy in the treatment of drug-resistant tuberculosis. This article provides an in-depth and critical analysis of various strategies employed by the drug-resistant M. tuberculosis to escape the host immune response. This bacterium persists in the host for a longer period of time and leads to the development of tuberculosis infection. Furthermore, we also discussed the new targets for the effective treatment of drug-resistant tuberculosis.

The profile of drug resistance of Mycobacterium tuberculosis and clinical features of extrapulmonary resistant tuberculosis among children living in Lviv region, Ukraine

Pediatric tuberculosis (TB) is a serious infectious disease that affects many children worldwide and is more likely to be extrapulmonary than adult TB. The purpose - to analyze the profile of drug resistance of Mycobacterium tuberculosis and clinical features of extrapulmonary resistant (EPR) TB among children from Lviv region, Ukraine. Materials and methods. We analyzed all cases of EPR TB (n=23) and extrapulmonary sensitive (EPS) TB (n=24) among 478 medical charts of children, who were hospitalized in the Lviv Anti-TB hospital during 2013-2020. Results. It was found out that EPR TB was diagnosed significantly more often at the age of 1 year and up to 3 years old than EPS TB and significantly less often - among children aged 4-7 years. The children with EPR TB were significantly more likely to live in rural areas and they were significantly more likely to be from families with less than 2 children, compared to EPS TB. The children with EPR TB were more often diagnosed with meningeal and central nervous system (CNS) TB, less often - with TB of the bones and joints, only they had TB of the intestine, compared to EPS TB. Miliary pulmonary TB and the predominance of bilateral process were more common at EPR TB. Among children with EPR TB, rifampicin-resistant TB was significantly more common found than the risk of multidrug-resistant TB (MDR-TB) and monoresistant TB. The resistance profile of MDR-TB showed that 17.4% are resistant to the combination of HR (H-isoniazid, R-rifampicin), 8.6% - to HRES (E-ethambutol, S-streptomycin), 4.3% - to НRS. Among 43.5% of children with EPR TB the contact with a TB patient was not established. At the same time, only a third of children who had came into contact with bacterial exсretors were under dispensary observation and only about 9% received chemoprophylaxis. Conclusions. In order to prevent the development of EPR TB, it is necessary to improve TB prevention measures among the most vulnerable segments of the population. The research was carried out in accordance with the principles of the Helsinki Declaration. The study protocol was approved by the Local Ethics Committee of all participating institutions. The informed consent of the patient was obtained for conducting the studies. No conflict of interests was declared by the authors.

Isoxazole carboxylic acid methyl ester-based urea and thiourea derivatives as promising antitubercular agents.

In our continued efforts to find potential chemotherapeutics active against drug-resistant (DR) Mycobacterium tuberculosis (Mtb), causative agent of Tuberculosis (TB) and to curb the current burdensome treatment regimen, herein we describe the synthesis and biological evaluation of urea and thiourea variants of 5-phenyl-3-isoxazolecarboxylic acid methyl esters as promising anti-TB agent. Majority of the tested compounds displayed potent in vitro activity not only against drug-susceptible (DS) Mtb H37Rv but also against drug-resistant (DR) Mtb. Cell viability test against Vero cells deemed these compounds devoid of significant toxicity. 3,4-Dichlorophenyl derivative (MIC 0.25 µg/mL) and 4-chlorophenyl congener (MIC 1 µg/mL) among urea and thiourea libraries respectively exhibited optimum potency. Lead optimization resulted in the identification of 1,4-linked analogue of 3,4-dichlorophenyl urea derivative demonstrating improved selectivity. Further, in silico study complemented with previously proposed prodrug like attributes of isoxazole esters. Taken together, this molecular hybridization approach presents a new chemotype having potential to be translated into an alternate anti-Mtb agent. Graphical abstract Supplementary InformationThe online version contains supplementary material available at 10.1007/s11030-022-10543-0.

Expression analysis of efflux gene Rv1634 in drug resistance Mycobacterium tuberculosis isolates

Drug resistant tuberculosis (DR-TB) is a serious health issue worldwide and a number of mechanisms have been suggested which are involved in developing resistance in Mycobacterium tuberculosis to anti-tuberculosis drugs. In the Indian perspective, the pattern and prevalence of drug-resistant tuberculosis (DR-TB) are heterogeneous across regions and settings. Notably, isoniazid (INH), rifampicin (RIF) and ethambutol (EMB) are the most effective drugs in tuberculosis therapy. Understanding the pattern of resistance to these drugs in the Indian population is essential in quick diagnosis of multidrug-resistant (MDR) tuberculosis and extensive DR-TB (XDR-TB). A total of 36 Mycobacterium isolates from the North Indian population were exposed and tested for drug susceptibility with INH, RIF and EMB in vitro and results showed that 25, 24 and 24 isolates were resistant to INH, RIF and EMB respectively. Gene expression analysis revealed that approximately 50% of isolates under the stress of these three anti-tuberculosis drugs showed overexpression of efflux gene Rv1634. Moreover, overexpression of gene Rv1634 contributes to the emergence of drug resistance in M. tuberculosis and inhibition of Rv1634 along with other efflux pump genes and may provide a promising strategy for improving tuberculosis treatment outcomes in North India.

Tools to Alleviate the Drug Resistance in Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb), an acid-fast bacillus that causes Tuberculosis (TB), is a pathogen that caused 1.5 million deaths in 2020. As per WHO estimates, another 4.1 million people are suffering from latent TB, either asymptomatic or not diagnosed, and the frequency of drug resistance is increasing due to intrinsically linked factors from both host and bacterium. For instance, poor access to TB diagnosis and reduced treatment in the era of the COVID-19 pandemic has resulted in more TB deaths and an 18% reduction in newly diagnosed cases of TB. Additionally, the detection of Mtb isolates exhibiting resistance to multiple drugs (MDR, XDR, and TDR) has complicated the scenario in the pathogen’s favour. Moreover, the conventional methods to detect drug resistance may miss mutations, making it challenging to decide on the treatment regimen. However, owing to collaborative initiatives, the last two decades have witnessed several advancements in both the detection methods and drug discovery against drug-resistant isolates. The majority of them belong to nucleic acid detection techniques. In this review, we highlight and summarize the molecular mechanism underlying drug resistance in Mtb, the recent advancements in resistance detection methods, and the newer drugs used against drug-resistant TB.

Unraveling the mechanisms of intrinsic drug resistance in Mycobacterium tuberculosis.

Tuberculosis (TB) is among the most difficult infections to treat, requiring several months of multidrug therapy to produce a durable cure. The reasons necessitating long treatment times are complex and multifactorial. However, one major difficulty of treating TB is the resistance of the infecting bacterium, Mycobacterium tuberculosis (Mtb), to many distinct classes of antimicrobials. This review will focus on the major gaps in our understanding of intrinsic drug resistance in Mtb and how functional and chemical-genetics can help close those gaps. A better understanding of intrinsic drug resistance will help lay the foundation for strategies to disarm and circumvent these mechanisms to develop more potent antitubercular therapies.

The Role of Phosphorylation and Acylation in the Regulation of Drug Resistance in Mycobacterium tuberculosis.

Tuberculosis is a chronic and lethal infectious disease caused by Mycobacterium tuberculosis. In previous decades, most studies in this area focused on the pathogenesis and drug targets for disease treatments. However, the emergence of drug-resistant strains has increased the difficulty of clinical trials over time. Now, more post-translational modified proteins in Mycobacterium tuberculosis have been discovered. Evidence suggests that these proteins have the ability to influence tuberculosis drug resistance. Hence, this paper systematically summarizes updated research on the impacts of protein acylation and phosphorylation on the acquisition of drug resistance in Mycobacterium tuberculosis through acylation and phosphorylation protein regulating processes. This provides us with a better understanding of the mechanism of antituberculosis drugs and may contribute to a reduction the harm that tuberculosis brings to society, as well as aiding in the discovery of new drug targets and therapeutic regimen adjustments in the future.

Mycobacterium tuberculosis Drug Resistance in Ethiopia: An Updated Systematic Review and Meta-Analysis.

Background: Tuberculosis (TB) remains a significant global public health issue, despite advances in diagnostic technologies, substantial global efforts, and the availability of effective chemotherapies. Mycobacterium tuberculosis, a species of pathogenic bacteria resistant to currently available anti-TB drugs, is on the rise, threatening national and international TB-control efforts. This systematic review and meta-analysis aims to estimate the pooled prevalence of drug-resistant TB (DR-TB) in Ethiopia. Materials and Methods: A systematic literature search was undertaken using PubMed/MEDLINE, HINARI, the Web of Science, ScienceDirect electronic databases, and Google Scholar (1 January 2011 to 30 November 2020). After cleaning and sorting the records, the data were analyzed using STATA 11. The study outcomes revealed the weighted pooled prevalence of any anti-tuberculosis drug resistance, any isoniazid (INH) and rifampicin (RIF) resistance, monoresistance to INH and RIF, and multidrug-resistant TB (MDR-TB) in newly diagnosed and previously treated patients with TB. Results: A total of 24 studies with 18,908 patients with TB were included in the final analysis. The weighted pooled prevalence of any anti-TB drug resistance was 14.25% (95% confidence interval (CI): 7.05–21.44%)), whereas the pooled prevalence of any INH and RIF resistance was found in 15.62% (95%CI: 6.77–24.47%) and 9.75% (95%CI: 4.69–14.82%) of patients with TB, respectively. The pooled prevalence for INH and RIF-monoresistance was 6.23% (95%CI: 4.44–8.02%) and 2.33% (95%CI: 1.00–3.66%), respectively. MDR-TB was detected in 2.64% (95%CI: 1.46–3.82%) of newly diagnosed cases and 11.54% (95%CI: 2.12–20.96%) of retreated patients with TB, while the overall pooled prevalence of MDR-TB was 10.78% (95%CI: 4.74–16.83%). Conclusions: In Ethiopia, anti-tuberculosis drug resistance is widespread. The estimated pooled prevalence of INH and RIF-monoresistance rates were significantly higher in this review than in previous reports. Moreover, MDR-TB in newly diagnosed cases remained strong. Thus, early detection of TB cases, drug-resistance testing, proper and timely treatment, and diligent follow-up of TB patients all contribute to the improvement of DR-TB management and prevention. Besides this, we urge that a robust, routine laboratory-based drug-resistance surveillance system be implemented in the country.

Specific Profiles and Amplification of Resistance to Anti-tuberculosis Drugs in Tuberculosis Patients in the Republic of Karelia

The objective: to determine the frequency of resistance of Mycobacterium tuberculosis (MTB) to the most effective anti-tuberculosis drugs in tuberculosis patients in the Republic of Karelia, to characterize cases of amplification of MTB drug resistance during chemotherapy (CT).Subjects and Methods. MTB cultures of 1,137 tuberculosis patients were studied, the patients received chemotherapy from 2016 to 2021, among them 634 (55.8%) had multiple drug resistance (MDR). Among MBT cultures with MDR, there was a high frequency of MTB with extensive drug resistance (XDR) – 42.3% (268/634) including new cases. It provided a negative impact on the chemotherapy efficacy. Most often, amplification of MTB drug resistance was observed in patients treated as per regimen IV – 37/49 (75.5%) cases. MTB drug resistance amplified more often in the cases who were already resistant to 3 and more anti-tuberculosis drugs, 31/49 (63.3%). Most often, amplification was noted in aminoglycosides (Km, Am) and glycopeptide (Cm), (71.6% of cases). The high frequency of MTB drug resistance amplification from MDR up to XDR was due to the lack of rapid molecular genetic methods to test drug susceptibility to fluoroquinolones and aminoglycosides.

Evolution of Mycobacterium tuberculosis drug resistance in the genomic era.

Mycobacterium tuberculosis has acquired drug resistance to all drugs that have been used against it, including those only recently introduced into clinical practice. Compared to other bacteria, it has a well conserved genome due to its role as an obligate human pathogen that has adapted to a niche over five to ten thousand years. These features facilitate reconstruction and dating of M. tuberculosis phylogenies, giving key insights into how resistance has been acquired and spread globally. Resistance to each new drug has occurred within five to ten years of clinical use and has occurred even more rapidly with recently introduced drugs. In most cases, resistance-conferring mutations come with a fitness cost, but this can be overcome by compensatory mutations which restore fitness to that of wild-type bacteria. It is likely that M. tuberculosis acquires drug resistance while maintaining limited genomic variability due the generation of low frequency within-host variation, combined with ongoing purifying selection causing loss of variants without a clear fitness advantage. However, variants that do confer an advantage, such as drug resistance, can increase in prevalence amongst all bacteria within a host and become the dominant clone. These resistant strains can then be transmitted leading to primary drug resistant infection in a new host. As many countries move towards genomic methods for diagnosis of M. tuberculosis infection and drug resistance, it is important to be aware of the implications for the evolution of resistance. Currently, understanding of resistance-conferring mutations is incomplete, and some targeted genetic diagnostics create their own selective pressures. We discuss an example where a rifampicin resistance-conferring mutation which was not routinely covered by standard testing became dominant. Finally, resistance to new drugs such as bedaquiline and delamanid is caused by individually rare mutations occurring across a large mutational genomic target that have been detected over a short time, and do not provide statistical power for genotype-phenotype correlation – in contrast to longer-established drugs that form the backbone of drug-sensitive antituberculosis therapy. Therefore, we need a different approach to identify resistance-conferring mutations of new drugs before their resistance becomes widespread, abrogating their usefulness.

The evolving biology of Mycobacterium tuberculosis drug resistance.

Tuberculosis, caused by Mycobacterium tuberculosis (Mtb) is an ancient disease that has remained a leading cause of infectious death. Mtb has evolved drug resistance to every antibiotic regimen ever introduced, greatly complicating treatment, lowering rates of cure and menacing TB control in parts of the world. As technology has advanced, our understanding of antimicrobial resistance has improved, and our models of the phenomenon have evolved. In this review, we focus on recent research progress that supports an updated model for the evolution of drug resistance in Mtb. We highlight the contribution of drug tolerance on the path to resistance, and the influence of heterogeneity on tolerance. Resistance is likely to remain an issue for as long as drugs are needed to treat TB. However, with technology driving new insights and careful management of newly developed resources, antimicrobial resistance need not continue to threaten global progress against TB, as it has done for decades.

Increased Susceptibility of Mycobacterium tuberculosis to Ethionamide by Expressing PPs-Induced Rv0560c

Tuberculosis, an infectious disease, is one of the leading causes of death worldwide. Drug-resistant tuberculosis exacerbates its threat. Despite long-term and costly treatment with second-line drugs, treatment failure rates and mortality remain high. Therefore, new strategies for developing new drugs and improving the efficiency of existing drug treatments are urgently needed. Our research team reported that PPs, a new class of potential anti-tuberculosis drug candidates, can inhibit the growth of drug-resistant Mycobacterium tuberculosis. Here, we report a synergistic effect of PPs with ethionamide (ETH), one of the second-line drugs, as a result of further research on PPs. While investigating gene expression changes based on microarray and 2DE (two-dimensional gel electrophoresis), it was found that PPs induced the greatest overexpression of Rv0560c in M. tuberculosis. Based on this result, a protein microarray using Rv0560c protein was performed, and it was confirmed that Rv0560c had the highest interaction with EthR, a repressor for EthA involved in activating ETH. Accordingly, a synergistic experiment was conducted under the hypothesis of increased susceptibility of ETH to M. tuberculosis by PPs. As a result, in the presence of 0.5× MIC PPs, ETH showed a growth inhibitory effect on drug-sensitive and -resistant M. tuberculosis even at a much lower concentration of about 10-fold than the original MIC of ETH. It is also suggested that the effect was due to the interaction between PPs and Rv2887, the repressor of Rv0560c. This effect was also confirmed in a mouse model of pulmonary tuberculosis, confirming the potential of PPs as a booster to enhance the susceptibility of M. tuberculosis to ETH in treating drug-resistant tuberculosis. However, more in-depth mechanistic studies and extensive animal and clinical trials are needed in the future.

Mycobacterium tuberculosis functional genetic diversity, altered drug sensitivity, and precision medicine.

In the face of the unrelenting global burden of tuberculosis (TB), antibiotics remain our most effective tools to save lives and control the spread of Mycobacterium tuberculosis (Mtb). However, we confront a dual challenge in our use of antibiotics: simplifying and shortening the TB drug regimen while also limiting the emergence and propagation of antibiotic resistance. This task is now more feasible due to the increasing availability of bacterial genomic data at or near the point of care. These resources create an opportunity to envision how integration of bacterial genetic determinants of antibiotic response into treatment algorithms might transform TB care. Historically, Mtb drug resistance studies focused on mutations in genes encoding antibiotic targets and the resulting increases in the minimal inhibitory concentrations (MICs) above a breakpoint value. But recent progress in elucidating the effects of functional genetic diversity in Mtb has revealed various genetic loci that are associated with drug phenotypes such as low-level MIC increases and tolerance which predict the development of resistance and treatment failure. As a result, we are now poised to advance precision medicine approaches in TB treatment. By incorporating information regarding Mtb genetic characteristics into the development of drug regimens, clinical care which tailors antibiotic treatment to maximize the likelihood of success has come into reach.

Identification of Concomitant Inhibitors against Glutamine Synthetase and Isocitrate Lyase in Mycobacterium tuberculosis from Natural Sources.

Tuberculosis (T.B.) is a disease that occurs due to infection by the bacterium, Mycobacterium tuberculosis (Mtb), which is responsible for millions of deaths every year. Due to the emergence of multidrug and extensive drug-resistant Mtb strains, there is an urgent need to develop more powerful drugs for inclusion in the current tuberculosis treatment regime. In this study, 1778 molecules from four medicinal plants, Azadirachta indica, Camellia sinensis, Adhatoda vasica, and Ginkgo biloba, were selected and docked against two chosen drug targets, namely, Glutamine Synthetase (G.S.) and Isocitrate Lyase (I.C.L.). Molecular Docking was performed using the Glide module of the Schrӧdinger suite to identify the best-performing ligands; the complexes formed by the best-performing ligands were further investigated for their binding stability via Molecular Dynamics Simulation of 100 ns. The present study suggests that Azadiradione from Azadirachta indica possesses the potential to inhibit Glutamine Synthetase and Isocitrate Lyase of M. tuberculosis concomitantly. The excellent docking score of the ligand and the stability of receptor-ligand complexes, coupled with the complete pharmacokinetic profile of Azadiradione, support the proposal of the small molecule, Azadiradione as a novel antitubercular agent. Further, wet lab analysis of Azadiradione may lead to the possible discovery of a novel antitubercular drug.

Highly Sensitive Detection of Isoniazid Heteroresistance in Mycobacterium Tuberculosis by Droplet Digital PCR.

The drug resistance of Mycobacterium tuberculosis constitutes a major public health threat. Existing approaches make it challenging to detect low levels of drug-resistant TB, also known as heteroresistance (HR), in a population. The recently found droplet digital PCR (ddPCR) is a sensitive method for determining the precise amount of nucleic acid in a sample. We used ddPCR to test the Mycobacterium tuberculosis heteroresistance because it delivers more exact quantitative data without the need for a reference curve. A TaqMan-MGB probe mutation detection assay was developed in order to determine the mutant and wild-type sequences of the isoniazid resistance katG (315) gene. We produced heteroresistant MTB combinations, which were subsequently identified by ddPCR, qPCR, and MeltPro/INH. In addition, 21 clinical sputum samples with positive smears were used to validate each method's capacity to determine HR in sputum. We discovered that ddPCR can detect mutant sequences in as few as 0.01% of a combination. DeepMelt TB/INH, which is less sensitive in comparison, cannot detect HR with high resolution and requires a mutation rate of 50% to identify. qPCR likewise has a high resolution of 0.02%, but unlike ddPCR, it cannot determine the exact number of mutations. Our assay is applicable to sputum as well. ddPCR found a katG 315 substitution in two sputums with extremely low values of HR (0.26% and 0.14%). In 21 samples of clinical sputum, the HR prevalence of INH was 9.5%. This work demonstrates that a well-designed ddPCR HR detection test can detect low levels of HR with high accuracy and consistency and gives new information for the clinical diagnosis of drug resistance.