Identification of a mycobacterial hydrazidase, an isoniazid-hydrolyzing enzyme.

There exists decades-old evidence that some mycobacteria, including Mycobacterium avium and Mycobacterium smegmatis, produce hydrazidase, an enzyme that can hydrolyze the first-line antitubercular agent isoniazid. Despite its importance as a potential resistance factor, no studies have attempted to reveal its identity. In this study, we aimed to isolate and identify M. smegmatis hydrazidase, characterize it, and evaluate its impact on isoniazid resistance. We determined the optimal condition under which M. smegmatis produced the highest amount of hydrazidase, purified the enzyme by column chromatography, and identified it by peptide mass fingerprinting. It was revealed to be PzaA, an enzyme known as pyrazinamidase/nicotinamidase whose physiological role remains unknown. The kinetic constants suggested that this amidase with broad substrate specificity prefers amides to hydrazides as a substrate. Notably, of the five tested compounds, including amides, only isoniazid served as an efficient inducer of pzaA transcription, as revealed by quantitative reverse transcription PCR. Moreover, high expression of PzaA was confirmed to be beneficial for the survival and growth of M. smegmatis in the presence of isoniazid. Thus, our findings suggest a possible role for PzaA, and other hydrazidases yet to be identified, as an intrinsic isoniazid resistance factor of mycobacteria.

The physiological effect of rimI/rimJ silencing by CRISPR interference in Mycobacterium smegmatis mc2155.

N-terminal acetylation of proteins is an important post-translational modification (PTM) found in eukaryotes and prokaryotes. In bacteria, N-terminal acetylation is suggested to play various regulatory roles related to protein stability, gene expression, stress response, and virulence; however, the mechanism of such response remains unclear. The proteins, namely RimI/RimJ, are involved in N-terminal acetylation in mycobacteria. In this study, we used CRISPR interference (CRISPRi) to silence rimI/rimJ in Mycobacterium smegmatis mc2155 to investigate the physiological effects of N-terminal acetylation in cell survival and stress response. Repeat analysis of growth curves in rich media and biofilm analysis in minimal media of various mutant strains and wild-type bacteria did not show significant differences that could be attributed to the rimI/rimJ silencing. However, total proteome and acetylome profiles varied significantly across mutants and wild-type strains, highlighting the role of RimI/RimJ in modulating levels of proprotein acetylation in the cellular milieu. Further, we observed a significant increase in the minimum inhibitory concentration (MIC) (from 64 to 1024 µg ml-1) for the drug isoniazid in rimI mutant strains. The increase in MIC value for the drug isoniazid in the mutant strains suggests the link between N-terminal acetylation and antibiotic resistance. The study highlights the utility of CRISPRi as a convenient tool to study the role of PTMs, such as acetylation in mycobacteria. It also identifies rimI/rimJ genes as necessary for managing cellular response against antibiotic stress. Further research would be required to decipher the potential of targeting acetylation to enhance the efficacy of existing antibiotics.

[Clinical value of the MeltPro MTB assays in detection of drug-resistant tuberculosis in paraffin-embedded tissues].

Objective: To evaluate the clinical value of the MeltPro MTB assays in the diagnosis of drug-resistant tuberculosis. Methods: A cross-sectional study design was used to retrospectively collect all 4 551 patients with confirmed tuberculosis between January 2018 and December 2019 at Beijing Chest Hospital, Capital Medical University. Phenotypic drug sensitivity test and GeneXpert MTB/RIF (hereafter referred to as "Xpert") assay were used as gold standards to analyze the accuracy of the probe melting curve method. The clinical value of this technique was also evaluated as a complementary method to conventional assays of drug resistance to increase the detective rate of drug-resistant tuberculosis. Results: By taking the phenotypic drug susceptibility test as the gold standard, the sensitivity of the MeltPro MTB assays to detect resistance to rifampicin, isoniazid, ethambutol and fluoroquinolone was 14/15, 95.7%(22/23), 2/4 and 8/9,respectively; and the specificity was 92.0%(115/125), 93.2%(109/117), 90.4%(123/136) and 93.9%(123/131),respectively; the overall concordance rate was 92.1%(95%CI:89.6%-94.1%),and the Kappa value of the consistency test was 0.63(95%CI:0.55-0.72).By taking the Xpert test results as the reference, the sensitivity of this technology to the detection of rifampicin resistance was 93.6%(44/47), the specificity was100%(310/310), the concordance rate was 99.2%(95%CI:97.6%-99.7%), and the Kappa value of the consistency test was 0.96(95%CI:0.93-0.99). The MeltPro MTB assays had been used in 4 551 confirmed patients; the proportion of patients who obtained effective drug resistance results increased from 83.3% to 87.8%(P<0.01); and detection rate of rifampicin, isoniazid, ethambutol, fluoroquinolone resistance, multidrug and pre-extensive drug resistance cases were increased by 3.2%, 14.7%, 22.2%, 13.7%, 11.2% and 12.5%, respectively. Conclusion: The MeltPro MTB assays show satisfactory accuracy in the diagnosis of drug-resistant tuberculosis. This molecular pathological test is an effective complementary method in improving test positivity of drug-resistant tuberculosis.

Diagnostic performance of the GenoType MTBDRplus VER 2.0 line probe assay for the detection of isoniazid resistant Mycobacterium tuberculosis in Ethiopia.

BACKGROUND: Isoniazid (INH) resistant Mycobacterium tuberculosis (Hr-TB) is the most common type of drug resistant TB, and is defined as M tuberculosis complex (MTBC) strains resistant to INH but susceptible to rifampicin (RIF). Resistance to INH precedes RIF resistance in almost all multidrug resistant TB (MDR-TB) cases, across all MTBC lineages and in all settings. Therefore, early detection of Hr-TB is critical to ensure rapid initiation of appropriate treatment, and to prevent progression to MDR-TB. We assessed the performance of the GenoType MTBDRplus VER 2.0 line probe assay (LPA) in detecting isoniazid resistance among MTBC clinical isolates. METHODS: A retrospective study was conducted among M. tuberculosis complex (MTBC) clinical isolates obtained from the third-round Ethiopian national drug resistance survey (DRS) conducted between August 2017 and December 2019. The sensitivity, specificity, positive predictive value, and negative predictive value of the GenoType MTBDRplus VER 2.0 LPA in detecting INH resistance were assessed and compared to phenotypic drug susceptibility testing (DST) using the Mycobacteria Growth Indicator Tube (MGIT) system. Fisher's exact test was performed to compare the performance of LPA between Hr-TB and MDR-TB isolates. RESULTS: A total of 137 MTBC isolates were included, of those 62 were Hr-TB, 35 were MDR-TB and 40 were INH susceptible. The sensitivity of the GenoType MTBDRplus VER 2.0 for detecting INH resistance was 77.4% (95% CI: 65.5-86.2) among Hr-TB isolates and 94.3% (95% CI: 80.4-99.4) among MDR-TB isolates (P = 0.04). The specificity of the GenoType MTBDRplus VER 2.0 for detecting INH resistance was 100% (95% CI: 89.6-100). The katG 315 mutation was observed in 71% (n = 44) of Hr-TB phenotypes and 94.3% (n = 33) of MDR-TB phenotypes. Mutation at position-15 of the inhA promoter region alone was detected in four (6.5%) Hr-TB isolates, and concomitantly with katG 315 mutation in one (2.9%) MDR-TB isolate. CONCLUSIONS: GenoType MTBDRplus VER 2.0 LPA demonstrated improved performance in detecting INH resistance among MDR-TB cases compared to Hr-TB cases. The katG315 mutation is the most common INH resistance conferring gene among Hr-TB and MDR-TB isolates. Additional INH resistance conferring mutations should be evaluated to improve the sensitivity of the GenoType MTBDRplus VER 2.0 for the detection of INH resistance among Hr-TB cases.

[Investigation of Efflux Pump Genes in Resistant Mycobacterium tuberculosis Complex Clinical Isolates Exposed to First Line Antituberculosis Drugs and Verapamil Combination]. / Birinci Seçenek Antitüberküloz Ilaçlara ve Verapamil Kombinasyonuna Maruz Birakilan Dirençli Mycobacterium tuberculosis Kompleks Klinik Izolatlarindaki Efluks Pompa Genlerinin Arastirilmasi.

Tuberculosis (TB) is caused by Mycobacterium tuberculosis, still one of the most common life-threatening infectious diseases worldwide. Although drug resistance in M.tuberculosis is mainly due to spontaneous chromosomal mutations in genes encoding drug target or drug activating enzymes, the resistance cannot be explained only by these mutations. Low permeability of the cell wall, drug inactivating enzymes and especially efflux pumps (EPs) are other mechanisms of drug resistance in mycobacteria. Efflux pump inhibitors (EPIs) binding to M.tuberculosis EPs were shown to inhibit efflux of anti-TB drugs, to enhance M.tuberculosis killing, to reduce drug resistance and to produce synergistic effects with first line anti-TB drugs. In this study, we aimed to determine the minimum inhibitory concentration (MIC) of first-line anti-TB drugs in the presence of verapamil (VER) and the expression of 21 putative EP genes belonged to the ATP-binding cassette (ABC), major facilitator superfamily (MFS) and resistance-nodulation-division (RND) families which might have caused the resistance in nine M.tuberculosis complex clinical isolates resistant to all of the first line anti-TB drugs. MIC values of the isolates were determined in 96-well U-bottom plates by the resazurin microtiter test (REMA) method based on the color change principle. According to the determined MIC values of each isolate, freshly grown cultures in Middlebrook 7H9 broth were exposed to first-line anti-TB drugs and MIC of first-line anti-TB drugs in the presence of VER (½ MIC) at 37°C for 48 hours for RNA extraction. The non-drug exposed cultures were used as control. Total RNA was extracted using the RNeasy Mini Kit (Qiagen GmbH, Hilden, Germany) and then treated with DNase I (Thermo Fischer Scientific Inc., Waltham, MA). Complementary DNA (cDNA) from the extracted RNAs was synthesized with the "First strand cDNA synthesis kit" (Thermo Fischer Scientific Inc., Waltham, MA) using oligo primers. The expression levels of efflux pump genes by quantitative realtime polymerase chain reaction (qRt-PCR) were performed using the QuantiTect SYBR Green Rt-PCR Kit (Qiagen, Germany). The housekeeping sigma factor gene sigA (Rv2703) was used as internal control in qRt­PCR assays. Relative quantification of the clinical isolates was determined by the 2-∆∆Ct method by comparing the expression levels of efflux genes in cultures exposed to primary anti-TB drugs and VER with those of non-drug exposed cultures. MIC values of nine isolates by REMA method was determined between 32-512 µg/mL, 1-128 µg/mL, 2-32 µg/mL, 4-16 µg/mL and 15.62-250 µg/mL for streptomycin (SM), isoniazid (INH), rifampicin (RIF), ethambutol (EMB) and VER, respectively. In the presence of ½ MIC VER, it was determined that the MIC of SM decreased 2-32 fold in eight isolates, the MIC of INH decreased by 2-8 fold in nine isolates, the MIC of RIF decreased by 2-16 fold in eight isolates, and the MIC of EMB decreased 2-4 fold in only five isolates. There was an increase in the expression of Rv1273c, Rv1456c, Rv1457 and Rv1819 efflux pump genes from the ABC family, Rv1634 and Rv0842 from the MFS family and Rv3823 efflux from the RND family in isolates exposed to ½ MIC of first-line anti-TB drugs stress. Rv1456c and Rv1819 were found to be associated with SM resistance, Rv1273c with EMB resistance, Rv1457, Rv0842 and Rv3823 with both RIF and EMB resistance, and Rv1634 with INH, RIF and EMB resistance. It was determined that there was a decrease in the expression levels of eight efflux pump genes from the ABC family (Rv1456c, Rv1457c, Rv1458c, Rv0194, Rv1272c, Rv1686c, Rv1687c, Rv1819c), six from MFS family (Rv0842, Rv0849, Rv1634, Rv2265, Rv2456c, Rv0876c) and two from RND family (Rv0507, Rv0676c) in isolates exposed to MIC of first-line anti-TB drugs in the presence of VER (½ MIC). Further studies with clinical isolates are needed to investigate the EPIs that can be used in alternative therapy and to determine the contribution of EPs to the development of resistance due to the increasing TB resistance.

An analytic feasibility study of the BD MAX™ MDR-TB assay for testing of non-sputum specimens for detection of the Mycobacterium tuberculosis complex (MTBC) and isoniazid (INH) and rifampin (RIF) resistance.

Rapid diagnosis of tuberculosis and drug resistance in extrapulmonary specimens can be challenging. The BD MAX™ multidrug resistant (MDR)-TB assay (BD MAX™) has demonstrated high sensitivity and specificity for the detection of the Mycobacterium tuberculosis complex (MTBC) as well as resistance to INH and Rifampin (RIF) in pulmonary specimens but has not been rigorously assessed in extrapulmonary samples. We evaluated the diagnostic accuracy of the BD MAX™ assay for the detection of MTBC and drug resistance in extrapulmonary specimens spiked with MTBC from the Johns Hopkins strain collection. A total of 1083 tests were performed across multiple sample types, with an overall percent agreement of 94.8% (795/839) for detection of MTBC and 99% (379/383) and 96.4% (323/335) for determination of INH and RIF resistance-conferring mutations, respectively. The BD MAX™ assay provides same day detection of MTBC and drug-resistance results and could be a beneficial diagnostic test in extrapulmonary sample types.

The past, present and future of tuberculosis treatment.

Tuberculosis (TB) is an ancient infectious disease. Before the availability of effective drug therapy, it had high morbidity and mortality. In the past 100 years, the discovery of revolutionary anti-TB drugs such as streptomycin, isoniazid, pyrazinamide, ethambutol and rifampicin, along with drug combination treatment, has greatly improved TB control globally. As anti-TB drugs were widely used, multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains of Mycobacterium tuberculosis emerged due to acquired genetic mutations, and this now presents a major problem for effective treatment. Genes associated with drug resistance have been identified, including katG mutations in isoniazid resistance, rpoB mutations in rifampin resistance, pncA mutations in pyrazinamide resistance, and gyrA mutations in quinolone resistance. The major mechanisms of drug resistance include loss of enzyme activity in prodrug activation, drug target alteration, overexpression of drug target, and overexpression of the efflux pump. During the disease process, Mycobacterium tuberculosis may reside in different microenvironments where it is expose to acidic pH, low oxygen, reactive oxygen species and anti-TB drugs, which can facilitate the development of non-replicating persisters and promote bacterial survival. The mechanisms of persister formation may include toxin-antitoxin (TA) modules, DNA protection and repair, protein degradation such as trans-translation, efflux, and altered metabolism. In recent years, the use of new anti-TB drugs, repurposed drugs, and their drug combinations has greatly improved treatment outcomes in patients with both drug-susceptible TB and MDR/XDR-TB. The importance of developing more effective drugs targeting persisters of Mycobacterium tuberculosis is emphasized. In addition, host-directed therapeutics using both conventional drugs and herbal medicines for more effective TB treatment should also be explored. In this article, we review historical aspects of the research on anti-TB drugs and discuss the current understanding and treatments of drug resistant and persistent tuberculosis to inform future therapeutic development.

Synthesis, antitubercular evaluation, and molecular docking studies of hybrid pyridinium salts derived from isoniazid.

In the quest to develop potent inhibitors for Mycobacterium tuberculosis, novel isoniazid-based pyridinium salts were designed, synthesized, and tested for their antimycobacterial activities against the H37 Rv strain of Mycobacterium tuberculosis using rifampicin as a standard. The pyridinium salts 4k, 4l, and 7d showed exceptional antimycobacterial activities with MIC90 at 1 µg/mL. The in vitro cytotoxicity and pharmacokinetics profiles of these compounds were established for the identification of a lead molecule using in vivo efficacy proof-of-concept studies and found that the lead compound 4k possesses LC50 value at 25 µg/mL. The in vitro antimycobacterial activity results were further supported by in silico studies with good binding affinities ranging from -9.8 to -11.6 kcal/mol for 4k, 4l, and 7d with the target oxidoreductase DprE1 enzyme. These results demonstrate that pyridinium salts derived from isoniazid can be a potentially promising pharmacophore for the development of novel antitubercular candidates.

Clinical Evaluation of the XDR-LFC Assay for the Molecular Detection of Isoniazid, Rifampin, Fluoroquinolone, Kanamycin, Capreomycin, and Amikacin Drug Resistance in a Prospective Cohort.

While the goal of universal drug susceptibility testing has been a key component of the WHO End TB Strategy, in practice, this remains inaccessible to many. Rapid molecular tests for tuberculosis (TB) and antituberculosis drug resistance could significantly improve access to testing. In this study, we evaluated the accuracy of the Akonni Biosystems XDR-TB (extensively drug-resistant TB) TruArray and lateral-flow-cell (XDR-LFC) assay (Akonni Biosystems, Inc., Frederick, MD, USA), a novel assay that detects mutations in seven genes associated with resistance to antituberculosis drugs: katG, the inhA promoter, and the ahpC promoter for isoniazid; rpoB for rifampin; gyrA for fluoroquinolones; rrs and the eis promoter for kanamycin; and rrs for capreomycin and amikacin. We evaluated assay performance using direct sputum samples from 566 participants recruited in a prospective cohort in Moldova over 2 years. The sensitivity and specificity against the phenotypic reference were both 100% for isoniazid, 99.2% and 97.9% for rifampin, 84.8% and 99.1% for fluoroquinolones, 87.0% and 84.1% for kanamycin, 54.3% and 100% for capreomycin, and 79.2% and 100% for amikacin, respectively. Whole-genome sequencing data for a subsample of 272 isolates showed 95 to 99% concordance with the XDR-LFC-reported suspected mutations. The XDR-LFC assay demonstrated a high level of accuracy for multiple drugs and met the WHO's minimum target product profile criteria for isoniazid and rifampin, while the sensitivity for fluoroquinolones and amikacin fell below target thresholds, likely due to the absence of a gyrB target in the assay. With optimization, the XDR-LFC shows promise as a novel near-patient technology to rapidly diagnose drug-resistant tuberculosis.

A host blood transcriptional signature differentiates multi-drug/rifampin-resistant tuberculosis (MDR/RR-TB) from drug susceptible tuberculosis: a pilot study.

BACKGROUND: Tuberculosis (TB), caused by the bacterium Mycobacterium tuberculosis is one of the top thirteen causes of death worldwide. The major challenge to control TB is the emergence of drug-resistant tuberculosis (DR-TB); specifically, multi-drug resistant TB which are resistant to the most potent drugs; rifampin and isoniazid. Owing to the inconsistencies of the current diagnostic methods, a single test cannot identify the whole spectrum of DR-TB associated mutations. Recently, host blood transcriptomics has gained attention as a promising technique that develops disease-specific RNA signatures/biomarkers. However, studies on host transcriptomics infected with DR-TB is limited. Herein, we intended to identify genes/pathways that are differentially expressed in multi-drug/rifampin resistant TB (MDR/RR-TB) in contrast to drug susceptible TB. METHOD AND RESULTS: We conducted blood RNA sequencing of 10 pulmonary TB patients (4; drug susceptible and 6; DR-TB) and 55 genes that were differentially expressed in MDR/RR-TB from drug-susceptible/mono-resistant TB were identified. CD300LD, MYL9, VAMP5, CARD17, CLEC2B, GBP6, BATF2, ETV7, IFI27 and FCGR1CP were found to be upregulated in MDR/RR-TB in all comparisons, among which CLEC2B and CD300LD were not previously linked to TB. In comparison pathway analysis, interferon alpha/gamma response was upregulated while Wnt/beta catenin signaling, lysosome, microtubule nucleation and notch signaling were downregulated. CONCLUSION: Up/down-regulation of immunity related genes/pathways speculate the collective effect of hosts' attempt to fight against continuously multiplying DR-TB bacteria and the bacterial factors to fight against the host defense. The identified genes/pathways could act as MDR/RR-TB biomarkers, hence, further research on their clinical use should be encouraged.

Management of Polydrug-Resistant Tuberculosis.

Background and Objectives: There is a lack of information regarding the effective duration of treatment necessary to prevent the development of acquired resistance when fluoroquinolones (FQ), and/or pyrazinamide (Z) resistance has occurred in patients with polydrug-resistant tuberculosis and isoniazid resistance. The management of these kinds of patients should be carried out in experienced centers according to drug susceptibility test results, clinical status of the patient and the extensity of the disease. Materials and Methods: We evaluated treatment regimens, treatment outcomes, and drug adverse effects in seven patients with polydrug-resistant tuberculosis, including those with Z and/or FQ resistance in a retrospective analysis Results: Regarding the patients with polydrug-resistant tuberculosis in addition to isoniazid (H) resistance, three had Z, two had FQ, and the remaining two had both Z and FQ resistance. In the intensive phase of the treatment, the patients were given at least four drugs according to drug susceptibility tests, and at least three drugs in the continuation phase. The duration of treatment was 9-12 months. Two of the patients were foreign nationals, and could not be followed up with due to returning to their home countries. Regarding the remaining five patients, three of them were terminated as they completed treatment, and two as cured. No recurrence was observed in the first year of the treatment. The most common, and serious drug side effect was seen for amikacin. Conclusions: In patients with polydrug-resistant TB, if Z and/or FQ resistance is detected in addition to H resistance, the treatment of these patients should be conducted on a case-by-case basis, taking into account the patient's resistance pattern, clinical condition, and disease prognosis. Close monitoring of the side effects will increase the success rate of the treatment.

Study of drug interaction, mutant frequency and mutant prevention concentration of DFMBT against Mycobacterium tuberculosis H37RV.

In the present study 7,7-Dimethyl-4-(4-trifluoromethyl-phenylamino)-2,4,4a,6,7,8-hexahydro-benzo[d] [1,3]thiazin-5-one (DFMBT) was synthesized and evaluated for in vitro activity against Mycobacterium tuberculosis (M.tb) H37RV. Results demonstrated that at 64x MIC, DFMBT completely sterilized the TB culture from day 4 of the incubation whereas at 32 and 16x MIC, it sterilized the TB culture from day 8. The bacterial cultures were completely sterilized by DFMBT at 8x MIC from day 16 of incubation. DFMBT showed 1.5 µg/mL MIC value as compared to the standard anti-tuberculosis drugs using broth macro-dilution method. The MBC value of DFMBT was found to be 6.0 µg/mL whereas for INH, RIF, AMK and LVX the values were found to be 0.312, 0.156, 5.0 and 5.0 µg/mL, respectively. The DFMBT in combination with INH/RIF or AMK showed the ∑FIC value of 0.258, 0.252 and 0.453, respectively indicating synergistic interaction. Moreover, the value of ∑FIC for the combination of DFMBT with LVX was found to be 1.33 suggesting and additive interaction. The post antibiotic effect of DFMBT at 1x and 64x MIC was found to be 29.89 ± 10.12 and 158.75 ± 17.50 h, respectively. The DFMBT showed an MPC value of 150 µg/mL which was intermediate between INH and RIF. In summary, DFMBT exhibits bacteriostatic as well as bactericidal effect on Mycobacterium tuberculosis H37RV. It has synergistic interaction with INH, RIF and AMK anti-TB drugs, descent post antibiotic effect, mutation frequency and mutant prevention concentration. Thus, DFMBT may be developed as an effective agent as anti-TB compound.

Inhibition of mycobacteria proliferation in macrophages by low cisplatin concentration through phosphorylated p53-related apoptosis pathway.

BACKGROUND: Drug resistance is a prominent problem in the treatment of tuberculosis, so it is urgent to develop new anti- tuberculosis drugs. Here, we investigated the effects and mechanisms of cisplatin (DDP) on intracellular Mycobacterium smegmatis to tap the therapeutic potential of DDP in mycobacterial infection. RESULTS: Macrophages infected with Mycobacterium smegmatis were treated with DDP alone or combined with isoniazid or rifampicin. The results showed that the bacterial count in macrophages decreased significantly after DDP (≤ 6 µg/mL) treatment. When isoniazid or rifampicin was combined with DDP, the number of intracellular mycobacteria was also significantly lower than that of isoniazid or rifampicin alone. Apoptosis of infected cells increased after 24 h of DDP treatment, as shown by flow cytometry and transmission electron microscopy detection. Transcriptome sequencing showed that there were 1161 upregulated and 645 downregulated differentially expressed genes (DEGs) between the control group and DDP treatment group. A Trp53-centered protein interaction network was found based on the top 100 significant DEGs through STRING and Cytoscape software. The expression of phosphorylated p53, Bax, JAK, p38 MAPK and PI3K increased after DDP treatment, as shown by Western blot analysis. Inhibitors of JAK, PI3K or p38 MAPK inhibited the increase in cell apoptosis and the reduction in the intracellular bacterial count induced by DDP. The p53 promoter Kevetrin hydrochloride scavenges intracellular mycobacteria. If combined with DDP, Kevetrin hydrochloride could increase the effect of DDP on the elimination of intracellular mycobacteria. In conclusion, DDP at low concentrations could activate the JAK, p38 MAPK and PI3K pathways in infected macrophages, promote the phosphorylation of p53 protein, and increase the ratio of Bax to Bcl-2, leading to cell apoptosis, thus eliminating intracellular bacteria and reducing the spread of mycobacteria. CONCLUSION: DDP may be a new host-directed therapy for tuberculosis treatment, as well as the p53 promoter Kevetrin hydrochloride.

Large-scale genomic analysis of Mycobacterium tuberculosis reveals extent of target and compensatory mutations linked to multi-drug resistant tuberculosis.

Resistance to isoniazid (INH) and rifampicin (RIF) first-line drugs in Mycobacterium tuberculosis (Mtb), together called multi-drug resistance, threatens tuberculosis control. Resistance mutations in katG (for INH) and rpoB (RIF) genes often come with fitness costs. To overcome these costs, Mtb compensatory mutations have arisen in rpoC/rpoA (RIF) and ahpC (INH) loci. By leveraging the presence of known compensatory mutations, we aimed to detect novel resistance mutations occurring in INH and RIF target genes. Across ~ 32 k Mtb isolates with whole genome sequencing (WGS) data, there were 6262 (35.7%) with INH and 5435 (30.7%) with RIF phenotypic resistance. Known mutations in katG and rpoB explained ~ 99% of resistance. However, 188 (0.6%) isolates had ahpC compensatory mutations with no known resistance mutations in katG, leading to the identification of 31 putative resistance mutations in katG, each observed in at least 3 isolates. These putative katG mutations can co-occur with other INH variants (e.g., katG-Ser315Thr, fabG1 mutations). For RIF, there were no isolates with rpoC/rpoA compensatory mutations and unknown resistance mutations. Overall, using WGS data we identified putative resistance markers for INH that could be used for genotypic drug-resistance profiling. Establishing the complete repertoire of Mtb resistance mutations will assist the clinical management of tuberculosis.

One-pot green synthesis of chitosan biguanidine nanoparticles for targeting M. tuberculosis.

Tuberculosis (TB) is considered as one of the most fatal infectious diseases nowadays. Several traditional anti-tuberculosis drugs like isoniazid have been largely applied; however, they are associated with toxicity and poor anti-TB treatment. So, the fabrication of new alternative anti-TB drugs containing natural biopolymers for TB treatment has attracted great attention in recent years because of their remarkable features: biodegradability, biocompatibility and non-toxicity. Therefore, their medicine is very effective with low side effects compared with synthetic drugs. Our current work intends to engineer chitosan biguanidine (ChBG) nanoparticles as a new safe and high-efficient anti-TB drug using one-pot, green, cost-effective ionic gelation method. The chemical structure of as-formed materials was chemically confirmed using various analysis techniques: H-NMR, FTIR, SEM, and TEM. TEM results have proved the formation of uniformly well-distributed ChBG nanoparticles with a small particle size of ~38 nm. The inhibitory activity of these prepared nanoparticles was investigated against the growth of three different M. tuberculosis pathogens such as sensitive, MDR, and XDR, and in a comparison with the isoniazid drug as a standard anti-tuberculosis drug. The antituberculosis assay results showed that ChBG NPs attained MIC values of 0.48, 3.9, 7.81 µg/mL for inhibiting the growth of sensitive, MDR, and XDR M. tuberculosis pathogens compared to bare Ch NPs (15.63, 62.5 > 125 µg/mL) and the isoniazid drug (0.24, 0, 0 µg/mL), respectively. Moreover, cytotoxicity of the ChBG NPs was examined against normal lung cell lines (Wi38) and was found to have cell viability of 100 % with the concentration range of 0.48-7.81 µg/mL.

Green engineering of TMC-CMS nanoparticles decorated graphene sheets for targeting M. tuberculosis.

Our current work intends to primarily engineer a new type of antibacterial composite by preparing a highly biocompatible graphene sheet decorated with TMC-CMS IPNs nanoparticles utilizing one-pot, green, cost-effective ultrasonication approach. The microstructure of as-formed materials was chemically confirmed using various analytical techniques such as 1H-NMR, FTIR, UV/vis, SEM, and TEM. TEM data has proved the formation of uniformly distributed TCNPs on graphene surfaces with a small particle size of ~22 nm compared with that of pure nanoparticles (~30 nm). The inhibitory activity of these developed materials was examined against the growth of three different M. tuberculosis pathogens and in a comparison with the isoniazid drug as a standard anti-tuberculosis drug. The TCNPs@GRP composite attained MIC values of 0.98, 3.9, and 7.81 µg/mL for inhibiting the growth of sensitive, MDR, and XDR M. tuberculosis pathogens compared to the bare TCNPs (7.81, 31.25, >125 µg/mL) and the isoniazid drug (0.24, 0, 0 µg/mL), respectively. This reveals a considerable synergism in the antituberculosis activity between TCNPs and graphene nanosheets. Cytotoxicity of the TCNPs@GRP was examined against normal lung cell lines (WI38) and was found to have cell viability of 100% with the concentration range of 0.98-7.81 µg/mL.

Gallium and indium complexes with isoniazid-derived ligands: Interaction with biomolecules and biological activity against cancer cells and Mycobacterium tuberculosis.

Gallium and indium octahedral complexes with isoniazid derivative ligands were successfully prepared. The ligands, isonicotinoyl benzoylacetone (H2L1) and 4-chlorobenzoylacetone isonicotinoyl hydrazone (H2L2), and their respective coordination compounds with gallium and indium [GaL1(HL1)] (GaL1), [GaL2(HL2)] (GaL2), [InL1(HL1)] (InL1) and [InL2(HL2)] (InL2) were investigated by NMR, ESI-MS, UV-Vis, IR, single-crystal X-ray diffraction and elemental analysis. In vitro interaction studies with human serum albumin (HSA) evidenced a moderate affinity of all complexes with HSA through spontaneous hydrophobic interactions. The greatest suppression of HSA fluorescence was caused by GaL2 and InL2, which was associated to the higher lipophilicity of H2L2. In vitro interaction studies with CT-DNA indicated weak interactions of the biomolecule with all complexes. Cytotoxicity assays with MCF-7 (breast carcinoma), PC-3 (prostate carcinoma) and RWPE-1 (healthy human prostate epithelial) cell lines showed that complexes with H2L2 are more active and selective against MCF-7, with the greatest cytotoxicity observed for InL2 (IC50 = 10.34 ± 1.69 µM). H2L1 and H2L2 were labelled with gallium-67, and it was verified that 67GaL2 has a greater lipophilicity than 67GaL1, as well as higher stability in human serum or in the presence of apo-transferrin. Cellular uptake assays with 67GaL1 and 67GaL2 evidenced that the H2L2-containing radiocomplex has a higher accumulation in MCF-7 and PC-3 cells than the non-halogenated congener 67GaL1. The anti-Mycobacterium tuberculosis assays revealed that both ligands and metal complexes are potent growth inhibitors, with MIC90 (µg mL-1) values observed from 0.419 ± 0.05 to 1.378 ± 0.21.

Discovery of a LuxR-type regulator involved in isoniazid-dependent gene regulation in Mycobacterium smegmatis.

OBJECTIVE: Most non-tuberculous mycobacteria exhibit intrinsic resistance against the anti-tuberculosis drug isoniazid (INH). We previously found that a pyrazinamidase/nicotinamidase of Mycobacterium smegmatis, named PzaA, has an enzymatic activity to hydrolyze INH, which may contribute to intrinsic resistance. Furthermore, PzaA expression is strongly induced by INH under nitrogen-depleted conditions, although the precise mechanism of this phenomenon remains unclear. Here, we aimed to reveal the mechanism underlying the INH-dependent induction of PzaA using a transcriptomic approach. METHODS: RNA sequencing was performed to identify INH-inducible genes other than pzaA. 5' rapid amplification of cDNA ends analysis was employed to identify the transcription start sites of INH-induced transcription units. The function of a LuxR-like regulator gene (MSMEI_1050) found within the gene cluster containing pzaA was confirmed by gene deletion and complementation experiments involving INH hydrolysis assay and quantitative reverse transcription PCR. RESULTS: RNA sequencing revealed 23 genes that INH strongly induced under conditions of nitrogen depletion, 17 of which were in a gene cluster containing pzaA. This cluster comprised at least three transcription units, including a non-INH-inducible monocistronic unit containing MSMEI_1050. Deletion of this gene deprived M. smegmatis of the ability to respond to INH, and complementation restored this ability. CONCLUSIONS: MSMEI_1050 plays a key role in INH-dependent gene regulation. The precise mechanism of action is to be determined in future studies.

Ultrasensitive Detection of Multidrug-Resistant Mycobacterium tuberculosis Using SuperSelective Primer-Based Real-Time PCR Assays.

The emergence of drug-resistant tuberculosis is a significant global health issue. The presence of heteroresistant Mycobacterium tuberculosis is critical to developing fully drug-resistant tuberculosis cases. The currently available molecular techniques may detect one copy of mutant bacterial genomic DNA in the presence of about 1-1000 copies of wild-type M. tuberculosis DNA. To improve the limit of heteroresistance detection, we developed SuperSelective primer-based real-time PCR assays, which, by their unique assay design, enable selective and exponential amplification of selected point mutations in the presence of abundant wild-type DNA. We designed SuperSelective primers to detect genetic mutations associated with M. tuberculosis resistance to the anti-tuberculosis drugs isoniazid and rifampin. We evaluated the efficiency of our assay in detecting heteroresistant M. tuberculosis strains using genomic DNA isolated from laboratory strains and clinical isolates from the sputum of tuberculosis patients. Results show that our assays detected heteroresistant mutations with a specificity of 100% in a background of up to 104 copies of wild-type M. tuberculosis genomic DNA, corresponding to a detection limit of 0.01%. Therefore, the SuperSelective primer-based RT-PCR assay is an ultrasensitive tool that can efficiently diagnose heteroresistant tuberculosis in clinical specimens and contributes to understanding the drug resistance mechanisms. This approach can improve the management of antimicrobial resistance in tuberculosis and other infectious diseases.

Emergence of phenotypic and genotypic antimicrobial resistance in Mycobacterium tuberculosis.

Concentration dependency of phenotypic and genotypic isoniazid-rifampicin resistance emergence was investigated to obtain a mechanistic understanding on how anti-mycobacterial drugs facilitate the emergence of bacterial populations that survive throughout treatment. Using static kill curve experiments, observing two evolution cycles, it was demonstrated that rifampicin resistance was the result of non-specific mechanisms and not associated with accumulation of drug resistance encoding SNPs. Whereas, part of isoniazid resistance could be accounted for by accumulation of specific SNPs, which was concentration dependent. Using a Hollow Fibre Infection Model it was demonstrated that emergence of resistance did not occur at concentration-time profiles mimicking the granuloma. This study showed that disentangling and quantifying concentration dependent emergence of resistance provides an improved rational for drug and dose selection although further work to understand the underlying mechanisms is needed to improve the drug development pipeline.