A descriptive study on isoniazid resistance-associated mutations, clustering and treatment outcomes of drug-resistant tuberculosis in a high burden country.

PURPOSE: To describe katG and inhA mutations, clinical characteristics, treatment outcomes and clustering of drug-resistant tuberculosis (TB) in the State of São Paulo, southeast Brazil. METHODS: Mycobacterium tuberculosis isolates from patients diagnosed with drug-resistant TB were screened for mutations in katG and inhA genes by line probe assay and Sanger sequencing, and typed by IS6110-restriction fragment-length polymorphism for clustering assessment. Clinical, epidemiological and demographic data were obtained from surveillance information systems for TB. RESULTS: Among the 298 isolates studied, 127 (42.6%) were isoniazid-monoresistant, 36 (12.1%) polydrug-resistant, 93 (31.2%) MDR, 16 (5.4%) pre-extensively drug-resistant (pre-XDR), 9 (3%) extensively drug-resistant (XDR) and 17 (5.7%) susceptible after isoniazid retesting. The frequency of katG 315 mutations alone was higher in MDR isolates, while inhA promoter mutations alone were more common in isoniazid-monoresistant isolates. Twenty-six isolates phenotypically resistant to isoniazid had no mutations either in katG or inhA genes. The isolates with inhA mutations were found more frequently in clusters (75%) when compared to the isolates with katG 315 mutations (59.8%, p = 0.04). In our population, being 35-64 years old, presenting MDR-, pre-XDR- or XDR-TB and being a retreatment case were associated with unfavourable TB treatment outcomes. CONCLUSION: We found that katG and inhA mutations were not equally distributed between isoniazid-monoresistant and MDR isolates. In our population, clustering was higher for isolates with inhA mutations. Finally, unfavourable TB outcomes were associated with specific factors.

Antimicrobial drug resistant features of Mycobacterium tuberculosis associated with treatment failure.

Tuberculosis stands as a prominent cause of mortality in developing countries. The treatment of tuberculosis involves a complex procedure requiring the administration of a panel of at least four antimicrobial drugs for the duration of six months. The occurrence of treatment failure after the completion of a standard treatment course presents a serious medical problem. The purpose of this study was to evaluate antimicrobial drug resistant features of Mycobacterium tuberculosis associated with treatment failure. Additionally, it aimed to evaluate the effectiveness of second line drugs such as amikacin, linezolid, moxifloxacin, and the efflux pump inhibitor verapamil against M. tuberculosis isolates associated with treatment failure. We monitored 1200 tuberculosis patients who visited TB centres in Lahore and found that 64 of them were not cured after six months of treatment. Among the M. tuberculosis isolates recovered from the sputum of these 64 patients, 46 (71.9%) isolates were simultaneously resistant to rifampicin and isoniazid (MDR), and 30 (46.9%) isolates were resistant to pyrazinamide, Resistance to amikacin was detected in 17 (26,5%) isolates whereas resistance to moxifloxacin and linezolid was detected in 1 (1.5%) and 2 (3.1%) isolates respectively. Among MDR isolates, the additional resistance to pyrazinamide, amikacin, and linezolid was detected in 15(23.4%), 4(2.6%) and 1(1.56%) isolates respectively. One isolate simultaneously resistant to rifampicin, isoniazid, amikacin, pyrazinamide, and linezolid was also identified. In our investigations, the most frequently mutated amino acid in the treatment failure group was Serine 315 in katG. Three novel mutations were detected at codons 99, 149 and 154 in pncA which were associated with pyrazinamide resistance. The effect of verapamil on the minimum inhibitory concentration of isoniazid and rifampicin was observed in drug susceptible isolates but not in drug resistant isolates. Rifampicin and isoniazid enhanced the transcription of the efflux pump gene rv1258 in drug susceptible isolates collected from the treatment failure patients. Our findings emphasize a high prevalence of MDR isolates linked primarily to drug exposure. Moreover, the use of amikacin as a second line drug may not be the most suitable choice in such cases.

Molecular diversity of Mycobacterium tuberculosis isolates in Treatment-experienced Patients from Andhra Pradesh, India.

INTRODUCTION: To get a comprehensive idea about the transmission and epidemiology of TB globally and locally, the use of molecular typing methods has become imperative not only for understanding genetic diversity but also the population structure of Mycobacterium tuberculosis complex (MTBC). We aimed to investigate the drug resistance pattern and genetic diversity of MTBC among previously treated patients with sputum smear-positive pulmonary tuberculosis in a South Indian population. METHODOLOGY: 104 patients with sputum smear positivity and who had previously undergone treatment were selected. Drug susceptibility testing, Spoligotyping, MIRU-VNTR, and SNP typing were performed. RESULTS: Mono-resistance to isoniazid 16 (15.38%) was the highest among all drugs. Out of 104 isolates, 24 (23%) isolates were classified as MDR strains. The distributions of most common lineages were: EAI3-Ind-20 (19.23%), EAI5-13 (12.50%), Beijing-12 (11.54%), CAS1-Delhi- 9 (8.65%), and 7 (6.73%) each of T-H37rv, Unknown and Orphan types. MIRU-VNTR-based analysis revealed that there are two major groups: CAS1-Delhi and Beijing groups. Out of 104 isolates, 82 belonged to well-defined lineages and 6 clusters, and the remaining 22 were singletons. SNP analysis showed no mutations associated with five sets of genes in 33 strains. CONCLUSIONS: The occurrence of 11.54% Beijing strains in South India is an important finding. High frequency of Isoniazid mono resistance noticed. Spoligotyping along with MIRU-VNTR and SNP typing is the best approach to the identification of strain lineages. No mutation with Antigen85C gene represents, can be used for vaccine candidates.

Deciphering the emerging role of phytocompounds: Implications in the management of drug-resistant tuberculosis and ATDs-induced hepatic damage.

Tuberculosis is a disease of poverty, discrimination, and socioeconomic burden. Epidemiological studies suggest that the mortality and incidence of tuberculosis are unacceptably higher worldwide. Genomic mutations in embCAB, embR, katG, inhA, ahpC, rpoB, pncA, rrs, rpsL, gyrA, gyrB, and ethR contribute to drug resistance reducing the susceptibility of Mycobacterium tuberculosis to many antibiotics. Additionally, treating tuberculosis with antibiotics also poses a serious risk of hepatotoxicity in the patient's body. Emerging data on drug-induced liver injury showed that anti-tuberculosis drugs remarkably altered levels of hepatotoxicity biomarkers. The review is an attempt to explore the anti-mycobacterial potential of selected, commonly available, and well-known phytocompounds and extracts of medicinal plants against strains of Mycobacterium tuberculosis. Many studies have demonstrated that phytocompounds such as flavonoids, alkaloids, terpenoids, and phenolic compounds have antibacterial action against Mycobacterium species, inhibiting the bacteria's growth and replication, and sometimes, causing cell death. Phytocompounds act by disrupting bacterial cell walls and membranes, reducing enzyme activity, and interfering with essential metabolic processes. The combination of these processes reduces the overall survivability of the bacteria. Moreover, several phytochemicals have synergistic effects with antibiotics routinely used to treat TB, improving their efficacy and decreasing the risk of resistance development. Interestingly, phytocompounds have been presented to reduce isoniazid- and ethambutol-induced hepatotoxicity by reversing serum levels of AST, ALP, ALT, bilirubin, MDA, urea, creatinine, and albumin to their normal range, leading to attenuation of inflammation and hepatic necrosis. As a result, phytochemicals represent a promising field of research for the development of new TB medicines.

Multi-drug resistant and rifampin-resistant tuberculosis in transplant recipients.

BACKGROUND: Management of multidrug-resistant (MDR) and rifampin-resistant (RR) tuberculosis is challenging. Data on transplant recipients is limited. We reviewed published literature to examine treatment choices, outcomes, and adverse effects from MDR-TB/RR-TB treatment in transplant recipients. METHODS: Multiple databases from inception to 12/2022 were reviewed using the keywords "drug-resistant TB" or "drug-resistant tuberculosis" or "multidrug-resistant TB" or "multidrug-resistant tuberculosis". MDR-TB was defined as resistance to isoniazid (H) and rifampin (R), and RR if resistant to rifampin alone. Cases without patient-level data and reports which did not describe treatment and/or outcomes for MDR-TB were excluded. RESULTS: A total of 12 patients (10 solid organ transplants and two hematopoietic cell transplants) were included. Of these, 11 were MDR-TB and one was RR-TB. Seven recipients were male. The median age was 41.5 (range 16-60) years. Pre-transplant evaluation for the majority (8/12, 66.7%) did not reveal a prior history of TB or TB treatment, but 9/12 were from TB intermediate or high-burden countries. Seven patients were initially treated with the quadruple first-line anti-TB regimen. Those who had early RR confirmation (5/12) via Xpert MTB/RIF assay were initiated on alternative therapies. Final regimens were individualized based on susceptibility profiles and tolerability. Adverse events were reported in seven recipients, including acute kidney injury (n = 3), cytopenias (n = 3), and jaundice (n = 2). Four recipients died, with two deaths attributable to TB. The remaining eight patients who survived had functioning allografts at the last follow-up. CONCLUSIONS: MDR-TB treatment in transplant recipients is associated with many complications. Xpert MTB/RIF detected RR early and guided early empiric therapy.

Isoniazid-N-acylhydrazones as promising compounds for the anti-tuberculosis treatment.

Tuberculosis (TB), a disease caused by Mycobacterium tuberculosis complex, still presents significant numbers of incidence and mortality, in addition to several cases of drug resistance. Resistance, especially to isoniazid, which is one of the main drugs used in the treatment, has increased. In this context, N-acylhydrazones derived from isoniazid have shown important anti-Mycobacterium tuberculosis activity. Hence, this work aimed to determine the anti-TB potential of 11 isoniazid-N-acylhydrazones (INH-acylhydrazones). For this purpose, the determination of minimum inhibitory concentration (MIC) against M. tuberculosis H37Rv and clinical isolates was carried out. Drug combination, minimum bactericidal concentration, cytotoxicity, and in silico parameters were also performed. INH-acylhydrazones (2), (8), and (9) had MIC for M. tuberculosis H37Rv similar to or lower than isoniazid, and bactericidal activity was observed. In addition, these compounds showed low cytotoxicity, with a selectivity index greater than 3,000. Interesting results were also obtained in the drug combination assay, with synergistic combinations with isoniazid, ethambutol, and rifampicin. In the in silico study, INH-acylhydrazones behaved similarly to INH, but with improvements in some aspects. Based on these findings, it is concluded that compounds (2), (8), and (9) are considered promising scaffolds and warrant further investigation for designing future antimicrobial drugs.

Drug resistance profile of Mycobacterium kansasii clinical isolates before and after 2-month empirical antimycobacterial treatment.

OBJECTIVES: Mycobacterium kansasii pulmonary disease is frequently misdiagnosed and treated as tuberculosis, especially in countries with high tuberculosis burden. This study aimed to investigate the drug resistance profile of M.kansasii in patients with M.kansasii pulmonary disease in Shanghai and to determine the variations in drug resistance after 2 months of antimycobacterial treatment. METHODS: All patients with a diagnosis of M.kansasii pulmonary disease from 2017 to 2019 in Shanghai were retrospectively analysed. Whole-genome sequencing was performed, and the minimum inhibitory concentration (MIC) to antimycobacterial drugs was measured using the broth microdilution method. RESULTS: In total, 191 patients had a diagnosis of M.kansasii pulmonary disease. Of them, 24.1% (46/191) had persistent positive culture after 2 months of antimycobacterial treatment. Whole-genome sequencing revealed that the 46 paired isolates had a difference of <17 single nucleotide polymorphisms, thus excluding the possibility of exogenous reinfection. More than 90% of the baseline isolates were sensitive to rifampin, clarithromycin, moxifloxacin, or amikacin, whereas a high resistance to ethambutol (118/191, 61.8%) and 4 µg/mL of isoniazid (32/191, 16.8%) were observed. Two isolates presented high resistance to rifamycin (i.e. a rifampin MIC of >8 µg/mL and a rifabutin MIC of 8 µg/mL) both containing the rpoB mutation (S454L). The increase of MIC to rifampin, ethambutol, and/or isoniazid was identified in 50.0% (23/46) of the patients. DISCUSSION: A high prevalence of innate resistance to ethambutol and isoniazid was observed among circulating M.kansasii clinical strains in Shanghai. The increase in drug resistance under empirical antimycobacterial treatment highlighted the urgency of definitive species identification before initiating treatment.

An effective nano drug delivery and combination therapy for the treatment of Tuberculosis.

Drug resistance in tuberculosis is exacerbating the threat this disease is posing to human beings. Antibiotics that were once effective against the causative agent, Mycobacterium tuberculosis (Mtb), are now no longer usable against multi- and extensively drug-resistant strains of this pathogen. To address this issue, new drug combinations and novel methods for targeted drug delivery could be of considerable value. In addition, studies have shown that the use of the antidepressant drug fluoxetine, a serotonin reuptake inhibitor, can be useful in the treatment of infectious diseases, including bacterial infections. In this study, an isoniazid and fluoxetine-conjugated multi-walled carbon nanotube nanofluid were designed to increase drug delivery efficiency alongside eliminating drug resistance in vitro. The prepared nanofluid was tested against Mtb. Expression levels of inhA and katG mRNAs were detected by Real-time PCR. ELISA was applied to measure levels of cytokine secretion (TNF-α, and IL-6) from infected macrophages treated with the nano delivery system. The results showed that these nano-drug delivery systems are effective for fluoxetine at far lower doses than for free drugs. Fluoxetine also has an additive effect on the effect of isoniazid, and their concomitant use in the delivery system can have significant effects in treating infection of all clinical strains of Mtb. In addition, it was found that the expression of isoniazid resistance genes, including inhA, katG, and the secretion of cytokines TNFα and IL6 under the influence of this drug delivery system is well regulated. It was shown that the drug conjugation can improve the antibacterial activity of them in all strains and these two drugs have an additive effect on each other both in free and conjugated forms. This nano-drug delivery method combined with host targeted molecules could be a game-changer in the development of a new generation of antibiotics that have high therapeutic efficiencies, low side effects, and the potential to overcome the problem of drug resistance.

Molecular detection of isoniazid monoresistance improves tuberculosis treatment: A retrospective cohort in France.

OBJECTIVES: Isoniazid-monoresistant tuberculosis (HR-TB) requires early diagnosis and adapted treatment to achieve optimal outcomes. The primary aim of the study was to assess the impact of the implementation of rapid diagnostic tests on HR-TB treatment in France. METHODS: We designed a retrospective multicentre study including consecutive HR-TB patients diagnosed in 2016 and 2017. Implementation of a molecular assay detecting isoniazid resistance directly on a clinical sample was recorded. The association between early implementation of such assays and adequate treatment was assessed by a multivariable Cox proportional hazards model. RESULTS: Overall, 99 HR-TB patients were included from 20 University Hospitals. Among all smear-positive HR-TB patients, only 26% beneficiated from early molecular HR detection. This detection was independently associated with shorter time to adequate treatment (HR = 2.0 [1.1-3.8], p = 0.03). CONCLUSION: In our study, molecular detection of HR on an initial sample was independently associated with earlier treatment adaptation.

Evaluating the clinical impact of routine whole genome sequencing in tuberculosis treatment decisions and the issue of isoniazid mono-resistance.

BACKGROUND: The UK has implemented routine use of whole genome sequencing (WGS) in TB diagnostics. The WHO recommends addition of a fluoroquinolone for isoniazid mono-resistance, so early detection may be of use. The aim of this study was to describe the clinical utility and impact of WGS on treatment decisions for TB in a low incidence high resource clinical setting. The clinical turnaround time (TAT) for WGS was analysed in comparison to TB PCR using Xpert MTB/RIF (Cepheid, Sunnyvale, CA) results where available and subsequent phenotypic drug susceptibility testing (DST) when required. METHODS: This was a retrospective analysis of TB cases from January 2018 to March 2019 in London. Susceptibility and TAT by WGS, phenotypic DST, TB PCR using Xpert MTB/RIF were correlated to drug changes in order to describe the utility of WGS on treatment decisions on isoniazid mono-resistance in a low incidence high resource setting. RESULTS: 189 TB cases were identified; median age 44 years (IQR 28-60), m:f ratio 112:77, 7 with HIV and 6 with previous TB. 80/189 cases had a positive culture and WGS result. 50/80 were fully sensitive to 1st line treatment on WGS, and the rest required additional DST. 20/80 cases required drug changes; 12 were defined by WGS: 8 cases had isoniazid mono-resistance, 2 had MDR-TB, 1 had isoniazid and pyrazinamide resistance and 1 had ethambutol resistance. The median TAT for positive culture was 16 days (IQR 12.5-20.5); for WGS was 35 days (IQR 29.5-38.75) and for subsequent DST was 86 days (IQR 69.5-96.75), resulting in non-WHO regimens for a median of 50.5 days (IQR 28.0-65.0). 9/12 has TB PCRs (Xpert MTB/RIF), with a median TAT of 1 day. CONCLUSION: WGS clearly has a substantial role in our routine UK clinical settings with faster turnaround times in comparison to phenotypic DST. However, the majority of treatment changes defined by WGS were related to isoniazid resistance and given the 1 month TAT for WGS, it would be preferable to identify isoniazid resistance more quickly. Therefore if resources allow, diagnostic pathways should be optimised by parallel use of WGS and new molecular tests to rapidly identify isoniazid resistance in addition to rifampicin resistance and to minimise delays in starting WHO isoniazid resistance treatment.

Dynamics of Extensive Drug Resistance Evolution of Mycobacterium tuberculosis in a Single Patient During 9 Years of Disease and Treatment.

Mycobacterium tuberculosis is one of the hardest to treat bacterial pathogens with a high capacity to develop antibiotic resistance by mutations. Here we have performed whole-genome sequencing of consecutive M. tuberculosis isolates obtained during 9 years from a patient with pulmonary tuberculosis. The infecting strain was isoniazid resistant and during treatment it stepwise accumulated resistance mutations to 8 additional antibiotics. Heteroresistance was common and subpopulations with up to 3 different resistance mutations to the same drug coexisted. Sweeps of different resistant clones dominated the population at different time points, always coupled to resistance mutations coinciding with changes in the treatment regimens. Resistance mutations were predominant and no hitch-hiking, compensatory, or virulence-increasing mutations were detected, showing that the dominant selection pressure was antibiotic treatment. The results highlight the dynamic nature of M. tuberculosis infection, population structure, and resistance evolution and the importance of rapid antibiotic susceptibility tests to battle this pathogen.

Evaluation of Five User-Friendly Whole Genome Sequencing Software for Mycobacterium tuberculosis in Clinical Application.

BACKGROUND: Whole genome sequencing (WGS) is an increasingly useful tool for tuberculosis (TB) diagnosis and disease management. In this study, we evaluated the utility of user-friendly WGS tools in reporting resistance profiles and identifying lineages of clinical TB isolates from South Korea. METHODS: Forty clinical samples from TB patients showing discrepancies between their rapid molecular and conventional drug susceptibility tests were used in this study. Among these clinical isolates, 37 strains were successfully evaluated via WGS software, using the GenTB, TB Profiler, PhyResSE, CASTB, and Mykrobe. RESULTS: More accurate and faster susceptibility results could be obtained with isoniazid than with rifampin. Using the phenotypic test as the gold standard, the isoniazid concordance rate between phenotypic drug susceptibility test (DST) and WGS (GenTB: 45.9%, TB profiler: 40.5%, PhyResSE: 40.5%, CASTB: 48.6%, and Mykrobe: 43.2%) was much higher than between phenotypic DST and rapid molecular genotypic DST (18.9%) among the 37 strains. In contrast, the rifampin concordance rate between phenotypic DST and WGS and that between phenotypic DST and rapid molecular genotypic DST was similar (81.1-89.2%). We also found novel mutations associated with INH in katG and ahpC gene region, not covered by the line probe assay. In addition, lineage analysis identified 81.1% of these samples as L2 East Asian lineage strains, and 18.9% as L4 Euro-American lineage strains. CONCLUSION: WGS may play a pivotal role in TB diagnosis and the detection of drug resistance, genetic diversity, and transmission dynamics in the near future because of its accuracy, speed, and extensibility.

Evaluation of Berberine as an Adjunct to TB Treatment.

Tuberculosis (TB) is the global health problem with the second highest number of deaths from a communicable disease after COVID-19. Although TB is curable, poor health infrastructure, long and grueling TB treatments have led to the spread of TB pandemic with alarmingly increasing multidrug-resistant (MDR)-TB prevalence. Alternative host modulating therapies can be employed to improve TB drug efficacies or dampen the exaggerated inflammatory responses to improve lung function. Here, we investigated the adjunct therapy of natural immune-modulatory compound berberine in C57BL/6 mouse model of pulmonary TB. Berberine treatment did not affect Mtb growth in axenic cultures; however, it showed increased bacterial killing in primary murine bone marrow-derived macrophages and human monocyte-derived macrophages. Ad libitum berberine administration was beneficial to the host in combination with rifampicin and isoniazid. Berberine adjunctive treatment resulted in decreased lung pathology with no additive or synergistic effects on bacterial burdens in mice. Lung immune cell flow cytometry analysis showed that adjunctive berberine treatment decreased neutrophil, CD11b+ dendritic cell and recruited interstitial macrophage numbers. Late onset of adjunctive berberine treatment resulted in a similar phenotype with consistently reduced numbers of neutrophils both in lungs and the spleen. Together, our results suggest that berberine can be supplemented as an immunomodulatory agent depending on the disease stage and inflammatory status of the host.

Retrospective Diagnostic Accuracy Study of Abbott RealTime MTB against Xpert MTB/RIF Ultra and Xpert MTB/RIF for the Diagnosis of Pulmonary Tuberculosis and Susceptibility to Rifampin and Isoniazid Treatment.

High-throughput centralized testing for tuberculosis (TB) and drug resistance is important, but comparative data are limited. In this retrospective cross-sectional study, participants were recruited from Johannesburg, South Africa, and Tbilisi, Georgia. The index tests, Abbott RealTime MTB (RT-MTB) and RealTime MTB RIF/INH (RT-MTB RIF/INH), were performed on specimens stored frozen for an extended period of time (beyond manufacturer-validated specifications) and compared to paired Xpert MTB/RIF Ultra (Xpert Ultra) and Xpert MTB/RIF (Xpert) results obtained with fresh specimens. The detection reference standard was the Mycobacterium tuberculosis complex culture, and for resistance detection, it was phenotypic drug susceptibility testing. The median age of 474 participants was 39 (interquartile range [IQR], 31 to 51) years. On decontaminated sputum, Xpert Ultra had a sensitivity of 91%, compared to 77% for RT-MTB, with a difference of +14% (95% confidence interval [CI], +9.2 to +21%; 18/127). On raw sputum, Xpert Ultra exhibited a sensitivity of 89% and Xpert one of 88%, compared to 80% for RT-MTB, exhibiting differences of +10% (95% CI, +3.3 to +18%; 9/93) and +8.6% (95% CI, +2.4 to +17%; 8/93), respectively. Specificity was ≥98% for all tests. All three tests showed high sensitivity and specificity for detection of rifampin resistance. Abbott assays may have lower sensitivity than Xpert and Xpert Ultra for TB detection but similar performance for detection of resistance. The differences in TB detection may be attributable to differences in testing of frozen (Abbott) versus fresh (Xpert) samples. Studies in compliance with manufacturer's instructions are required to compare performance. IMPORTANCE In 2019, 10 million people fell ill with tuberculosis (TB), of whom 1.4 million died. There are few comparative studies of diagnostic assays, particularly those aiming to be used in high-throughput laboratories. One such assay is the Abbott RealTime MTB (RT-MTB) and RealTime MTB RIF/INH (RT-MTB RIF/INH), which uses the m2000 platform already in use in many settings for HIV load testing and allows the diagnosis of TB and resistance to two first-line drugs, rifampin and isoniazid. Our study compared the RT-MTB and RT-MTB RIF/INH to the WHO-recommended Xpert MTB/RIF Ultra and Xpert MTB/RIF. The study is the largest comparative study to date and was performed independent of the manufacturer. The study results suggest that the Abbott RealTime MTB may have a lower sensitivity, but the study may have placed the Abbott test at a disadvantage by using frozen samples and comparing the results to those for fresh samples for the Xpert.

Bioenergetic Heterogeneity in Mycobacterium tuberculosis Residing in Different Subcellular Niches.

ATP/ADP depicts the bioenergetic state of Mycobacterium tuberculosis (Mtb). However, the metabolic state of Mtb during infection remains poorly defined due to the absence of appropriate tools. Perceval HR (PHR) was recently developed to measure intracellular ATP/ADP levels, but it cannot be employed in mycobacterial cells due to mycobacterial autofluorescence. Here, we reengineered the ATP/ADP sensor Perceval HR into PHR-mCherry to analyze ATP/ADP in fast- and slow-growing mycobacteria. ATP/ADP reporter strains were generated through the expression of PHR-mCherry. Using the Mtb reporter strain, we analyzed the changes in ATP/ADP levels in response to antimycobacterial agents. As expected, bedaquiline induced a decrease in ATP/ADP. Interestingly, the transcriptional inhibitor rifampicin led to the depletion of ATP/ADP levels, while the cell wall synthesis inhibitor isoniazid did not affect the ATP/ADP levels in Mtb. The usage of this probe revealed that Mtb faces depletion of ATP/ADP levels upon phagocytosis. Furthermore, we observed that the activation of macrophages with interferon gamma and lipopolysaccharides leads to metabolic stress in intracellular Mtb. Examination of the bioenergetics of mycobacteria residing in subvacuolar compartments of macrophages revealed that the bacilli residing in phagolysosomes and autophagosomes have significantly less ATP/ADP than the bacilli residing in phagosomes. These observations indicate that phagosomes represent a niche for metabolically active Mtb, while autophagosomes and phagolysosomes harbor metabolically quiescent bacilli. Interestingly, even in activated macrophages, Mtb residing in phagosomes remains metabolically active. We further observed that macrophage activation affects the metabolic state of intracellular Mtb through the trafficking of Mtb from phagosomes to autophagosomes and phagolysosomes. IMPORTANCE ATP/ADP levels guide bacterial cells, whether to replicate or to enter nonreplicating persistence. However, tools for measuring ATP/ADP levels with spatiotemporal resolution are lacking. Here, we describe a method for tracking ATP/ADP levels at the single-cell and population levels. Using this tool, we have demonstrated that the transcription inhibitor rifampicin induces metabolic stress. In contrast, the cell wall synthesis inhibitor isoniazid does not alter the metabolic state of the bacilli, suggesting that transcription is tightly intertwined with metabolism, while cell wall synthesis is not. Furthermore, we analyzed the metabolic state of mycobacteria residing in different compartments of macrophages. We observed that Mtb cells residing inside phagosomes have healthy ATP/ADP levels. In contrast, the bacteria residing inside phagolysosomes and autophagosomes face depletion of ATP. Interestingly, the activation of macrophages facilitates the trafficking of mycobacterial cells from metabolism-conducive phagosomes to metabolism-averse phagolysosomes and autophagosomes. We believe that this tool holds the key to the identification of inhibitors of mycobacterial metabolism.

Superfast Synthesis of Stabilized Silver Nanoparticles Using Aqueous Allium sativum (Garlic) Extract and Isoniazid Hydrazide Conjugates: Molecular Docking and In-Vitro Characterizations.

Green synthesis of silver nanoparticles (AgNPs) was synthesized from fresh garlic extract coupled with isoniazid hydrazide (INH), a commonly used antibiotic to treat tuberculosis. A molecular docking study conducted with the selected compounds compared with anthranilate phosphoribosyltransferase (trpD) from Mycobacterium tuberculosis. The aqueous extract of garlic was prepared and mixed with silver nitrate (AgNO3) solution for the superfast synthesis of stable AgNPs. INH was then conjugated with AgNPs at different ratios (v/v) to obtain stable INH-AgNPs conjugates (AgNCs). The resulting AgNCs characterized by FTIR spectra revealed the ultrafast formation of AgNPs (<5 s) and perfectly conjugated with INH. The shifting of λmax to longer wavelength, as found from UV spectral analysis, confirmed the formation of AgNCs, among which ideal formulations (F7, F10, and F13) have been pre-selected. The zeta particle size (PS) and the zeta potential (ZP) of AgNPs were found to be 145.3 ± 2.1 nm and -33.1 mV, respectively. These data were significantly different compared to that of AgNCs (160 ± 2.7 nm and -14.4 mV for F7; 208.9 ± 2.9 nm and -19.8 mV for F10; and 281.3 ± 3.6 nm and -19.5 mV for F13), most probably due to INH conjugation. The results of XRD, SEM and EDX confirmed the formation of AgNCs. From UV spectral analysis, EE of INH as 51.6 ± 5.21, 53.6 ± 6.88, and 70.01 ± 7.11 %, for F7, F10, and F13, respectively. The stability of the three formulations was confirmed in various physiological conditions. Drug was released in a sustainable fashion. Besides, from the preferred 23 compounds, five compounds namely Sativoside R2, Degalactotigonin, Proto-desgalactotigonin, Eruboside B and Sativoside R1 showed a better docking score than trpD, and therefore may help in promoting anti-tubercular activity.

Edible marine algae: a new source for anti-mycobacterial agents.

Tuberculosis is a dreaded disease, which causes innumerable death worldwide. The emergence of drug resistance strains makes the situation devastating. Therefore, for better management of public health, it is mandatory to search for new anti-mycobacterial agents. In this context, the current study investigated two edible marine algae, Ulva lactuca and Ulva intestinalis, for the probable source of new anti-mycobacterial agents. To test the anti-mycobacterial activity, alcoholic extracts of these two algae were spotted on the Mycobacterium smegmatis lawn. Upon incubation, clear zone was observed at the spots. It indicated that these two extracts have anti-mycobacterial activity. In addition, their anti-biofilm property was also tested. It was found that both the extracts inhibit the mycobacterial biofilm development as well as they can disperse the preformed mycobacterial biofilm. Since these two are capable of dispersing preformed mycobacterial biofilm, it is possible that in the presence of either of these two extracts, isoniazid and rifampicin can kill biofilm encapsulated mycobacterium in combinatorial therapy. Consistent with the hypothesis, rifampicin and isoniazid killed mycobacteria that were present in biofilm. Thus, these two extracts augment the activity of rifampicin and isoniazid upon biofilm dispersal. Moreover, treatment of different cell lines with these two extracts exhibited no or little cytotoxic effects. Thus, these two agents have the potential to be good therapeutic agents against mycobacterial diseases.

Multidrug-resistant tuberculosis in Imo State, Southeast, Nigeria.

BACKGROUND: Multidrug-resistant tuberculosis (MDR-TB) is a global health challenge. The emergence of MDR TB has contributed remarkably to the spread of tuberculosis and also poses a threat, which if not effectively addressed may wipe out the achievements of previous efforts in controlling tuberculosis. OBJECTIVE: This study was aimed at detecting MDR-TB among patients in a setting prevalent with tuberculosis and HIV in Southeast, Nigeria. METHOD: Sputum specimens collected from 740 suspected tuberculosis (TB) patients were screened for acid-fast bacilli (AFB). All the 111 AFB positive samples were subjected to culture on Lowenstein-Jensen (LJ) medium and Mycobacterium Growth Indicator Tube (MGIT) 960 TB system. The isolates were then confirmed as Mycobacterium tuberculosis using SD Bioline Rapid Diagnostic Tests before being subjected to drug susceptibility testing to first-line anti-TB drugs. MDR-TB was determined by isolates being resistant to both isoniazid and rifampicin. HIV testing was performed for participants included in the study using standard rapid diagnostic tests. RESULT: Out of the 111 AFB-positive sputum samples, 65 (58.6%) were culture-positive for Mycobacterium tuberculosis. MDR-TB was found in 2 ([3.1%] 95% CI = 0.0-7.3) of the culture-positive samples. The rate of TB and HIV coinfection was 7.7%. Maximum single-drug resistance was seen in ethambutol 12 ([18.5%] 95% CI = 9.0-27.9). CONCLUSION: The MDR-TB rate of 3.1% found in this study was relatively low and efforts should be intensified to keep it low.

A retrospective cohort study of isoniazid-resistant tuberculosis treatment outcomes and isoniazid resistance-associated mutations in eastern China from 2013 to 2018.

OBJECTIVES: The current situation of isoniazid-resistant, rifampicin-susceptible tuberculosis (Hr-TB) and associated genetic factors is not clear in China. METHODS: A retrospective cohort study was conducted from 2013 to 2018 in Jiangsu Province, China. Phenotypic Hr-TB were identified by drug susceptibility testing on Lowenstein-Jensen medium and using a Mycobacterium Growth Indicator Tube 960 (MGIT 960) system, and mutations in the katG 315 codon and inhA promoter nucleotides -8, -15 and -16 were determined by GenoType MTBDRplus and sequencing. All of the Hr-TB patients enrolled were followed up until June 2019. RESULTS: A total of 1416 smear-positive sputum samples were collected, of which 57 were excluded due to the presence of nontuberculous mycobacteria. Finally, 63/1359 (4.6%) were determined as Hr-TB. After follow-up, 11 Hr-TB patients (17.5%) showed an unfavourable outcome, of whom 5 (7.9%) relapsed, 4 (6.3%) had treatment failure and 2 (3.2%) died. A total of 52 isolates (82.5%) were detected with either katG 315 or inhA promoter nucleotide -8, -15 or -16 mutations, whereas no canonical mutations were found in 8 isolates (12.7%); 3 isolates failed in mutation detection. TB history was found to be associated with unfavourable outcomes for Hr-TB (odds ratio = 6.13, 95% confidence interval 1.05-35.82; P = 0.04). However, mutations in katG 315 and the inhA promoter region were not found to be associated with Hr-TB unfavourable outcomes (P = 0.15). CONCLUSION: Unfavourable outcomes for Hr-TB are serious in eastern China, especially for previously treated patients. Meanwhile, current genetic determination of Hr-TB is inadequate.

Synthesis, Characterization, and Biologic Activity of New Acyl Hydrazides and 1,3,4-Oxadiazole Derivatives.

Starting from isoniazid and carboxylic acids as precursors, thirteen new hydrazides and 1,3,4-oxadiazoles of 2-(4-substituted-phenoxymethyl)-benzoic acids were synthesized and characterized by appropriate means. Their biological properties were evaluated in terms of apoptosis, cell cycle blocking, and drug metabolism gene expression on HCT-8 and HT-29 cell lines. In vitro antimicrobial tests were performed by the microplate Alamar Blue assay for the anti-mycobacterial activities and an adapted agar disk diffusion technique for other non-tubercular bacterial strains. The best antibacterial activity (anti-Mycobacterium tuberculosis effects) was proved by 9. Compounds 7, 8, and 9 determined blocking of G1 phase. Compound 7 proved to be toxic, inducing apoptosis in 54% of cells after 72 h, an effect that can be predicted by the increased expression of mRNA caspases 3 and 7 after 24 h. The influence of compounds on gene expression of enzymes implicated in drug metabolism indicates that synthesized compounds could be metabolized via other pathways than NAT2, spanning adverse effects of isoniazid. Compound 9 had the best antibacterial activity, being used as a disinfectant agent. Compounds 7, 8, and 9, seemed to have antitumor potential. Further studies on the action mechanism of these compounds on the cell cycle may bring new information regarding their biological activity.