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Background: We aimed to evaluate the predictors for early and interim culture conversion within 2 months and 6 months of treatment in multidrug-resistant/rifampicin-resistant tuberculosis (MDR/RR-TB) patients in China., Methods: This study included adult MDR/RR-TB patients with a positive baseline sputum culture from 8 institutions located in different cities in China from May 2018 to January 2022. We mainly used logistic regression model to derive possible predictors of early and interim culture conversion., Results: A total of 813 patients were enrolled and 28.5% of them received multidrug-resistant treatment regimens containing bedaquiline. Of these, 362 (44.5%) patients experienced culture conversion within 2 months of treatment, and 649 (79.8%) within 6 months. The results of the multivariable logistic regression analysis revealed that acid-fast bacilli smear positive (adjusted odds ratio [aOR] = 1.637, 95% confidence interval [CI] = 1.197-2.238), cavities (aOR = 1.539, 95% CI = 1.132-2.092), bilateral disease (aOR = 1.638, 95% CI = 1.183-2.269), and viral hepatitis (aOR = 2.585, 95% CI = 1.189-5.622) were identified as risk factors for early culture un-conversion within 2 months of treatment. Additionally, smoking history (aOR = 2.197, 95% CI = 1.475-3.273), previous treatment for tuberculosis (aOR = 1.909, 95% CI = 1.282-2.844), bilateral disease (aOR = 2.201, 95% CI = 1.369-3.537), viral hepatitis (aOR = 2.329, 95% CI = 1.094-4.962) were identified as risk factors for interim culture un-conversion within 6 months of treatment, while patients with regimen containing bedaquiline (aOR = 0.310, 95% CI = 0.191-0.502) was a protective factor., Conclusions: A history of smoking, a baseline sputum AFB smear positive, lung cavities, bilateral disease, previous anti-tuberculosis treatment, or a comorbidity of viral hepatitis can be used as the predictors for early and interim culture un-conversion in MDR/RR-TB patients, while bedaquiline was a protective factor ., (© 2024. The Author(s).)

The Nix-TB clinical trial evaluated a new 6 month regimen containing three oral drugs; bedaquiline (B), pretomanid (Pa), and linezolid (L) (BPaL regimen) for the treatment of tuberculosis (TB). This regimen achieved remarkable results as almost 90% of the multidrug-resistant or extensively drug-resistant TB participants were cured but many patients also developed severe adverse events (AEs). The AEs were associated with the long-term administration of the protein synthesis inhibitor linezolid. Spectinamide 1599 is also a protein synthesis inhibitor of Mycobacterium tuberculosis with an excellent safety profile, but it lacks oral bioavailability. Here, we propose to replace L in the BPaL regimen with spectinamide (S) administered via inhalation and we demonstrate that inhaled spectinamide 1599, combined with BPa --BPaS regimen--has similar efficacy to that of the BPaL regimen while simultaneously avoiding the L-associated AEs. The BPaL and BPaS regimens were compared in the BALB/c and C3HeB/FeJ murine chronic TB efficacy models. After 4-weeks of treatment, both regimens promoted equivalent bactericidal effects in both TB murine models. However, treatment with BPaL resulted in significant weight loss and the complete blood count suggested the development of anemia. These effects were not similarly observed in mice treated with BPaS. BPaL and BPa, but not the BPaS treatment, also decreased myeloid to erythroid ratio suggesting the S in the BPaS regimen was able to recover this effect. Moreover, the BPaL also increased concentration of proinflammatory cytokines in bone marrow compared to mice receiving BPaS regimen. These combined data suggest that inhaled spectinamide 1599 combined with BPa is an effective TB regimen without L-associated AEs., Competing Interests: MZ, TD, AM, AW, CP, HL, JP, JP, MH, RL, JL, GR, AH, BM, MG No competing interests declared, (© 2024, Zohaib Ali et al.)

Despite known treatments, tuberculosis (TB) remains the world's top infectious killer, highlighting the pressing need for new drug regimens. To prioritize the most efficacious drugs for clinical testing, we previously developed a PK-PD translational platform with bacterial dynamics that reliably predicted short-term monotherapy outcomes in Phase IIa trials from preclinical mouse studies. In this study, we extended our platform to include PK-PD models that account for drug-drug interactions in combination regimens and bacterial regrowth in our bacterial dynamics model to predict cure at the end of treatment and relapse 6 months post-treatment. The Phase III STAND trial testing a new regimen comprised of pretomanid (Pa), moxifloxacin (M), and pyrazinamide (Z) (PaMZ) was suspended after a separate ongoing trial (NC-005) suggested that adding bedaquiline (B) to the PaMZ regimen would improve efficacy. To forecast if the addition of B would, indeed, benefit the PaMZ regimen, we applied an extended translational platform to both regimens. We predicted currently available short- and long-term clinical data well for drug combinations related to BPaMZ. We predicted the addition of B to PaMZ to shorten treatment duration by 2 months and to have similar bacteriological success to standard HRZE treatment (considering only treatment success but not withdrawal from side effects and other adverse events), both at the end of treatment for treatment efficacy and 6 months after treatment has ended in relapse prevention. Using BPaMZ as a case study, we have demonstrated our translational platform can predict Phase II and III outcomes prior to actual trials, allowing us to better prioritize the regimens most likely to succeed., Competing Interests: The authors declare no conflict of interest.

TBAJ-876, a second-generation diarylquinoline with greater antimycobacterial activity and a potentially better safety profile compared with bedaquiline, is under development for the treatment of drug-susceptible and drug-resistant tuberculosis (TB). A phase 1, first-in-human study of TBAJ-876, comprising a single-ascending dose (SAD) part including a food effect cohort, a multiple-ascending dose (MAD) part, and a relative bioavailability part of tablets versus oral suspension, was conducted on 137 healthy adults. A drug-drug interaction study was conducted on 28 healthy adults to evaluate the effects of TBAJ-876 on a cytochrome P450 3A4 substrate (midazolam) and a P-glycoprotein substrate (digoxin). TBAJ-876 was well-tolerated at single doses up to 800 mg and multiple doses up to 200 mg for 14 days. No deaths or serious adverse events occurred. No episodes of clinically significant prolongation of the QTc interval were observed. TBAJ-876 exposures were dose proportional in the SAD and MAD studies. TBAJ-876 exhibited multicompartmental pharmacokinetics (PK) with a long terminal half-life yielding quantifiable concentrations up to the longest follow-up of 10 weeks after a single dose and resulting in accumulation with multiple dosing. In the fed state, TBAJ-876 exposures approximately doubled with the tablet formulation, whereas M3 metabolite exposures decreased by approximately 20%. The relative bioavailability of TBAJ-876 was similar between tablets and the oral suspension at 100-mg doses. With co-administration of TBAJ-876, the AUC 0-inf of midazolam was unchanged and the C max was reduced by 14%; the AUC 0-last of digoxin was increased by 51%, and the C max was increased by 18%. These results support further investigation of TBAJ-876 for the treatment of tuberculosis., Competing Interests: The authors declare no conflict of interest.

The increasing clinical significance of Mycobacterium abscessus is owed to its innate high-level, broad-spectrum resistance to antibiotics and therefore rapidly evolves as an important human pathogen. This warrants the identification of novel targets for aiding the discovery of new drugs or drug combinations to treat M. abscessus infections. This study is inspired by the drug-hypersensitive profile of a mutant M. abscessus (U14) with transposon insertion in MAB_1915 . We validated the role of MAB_1915 in intrinsic drug resistance in M. abscessus by constructing a selectable marker-free in-frame deletion in MAB_1915 and complementing the mutant with the same or extended version of the gene and then followed by drug susceptibility testing. Judging by the putative function of MAB_1915, cell envelope permeability was studied by ethidium bromide accumulation assay and susceptibility testing against dyes and detergents. In this study, we established genetic evidence of the role of MAB_1915 in intrinsic resistance to rifampicin, rifabutin, linezolid, clarithromycin, vancomycin, and bedaquiline. Disruption of MAB_1915 has also been observed to cause a significant increase in cell envelope permeability in M. abscessus . Restoration of resistance is observed to depend on at least 27 base pairs upstream of the coding DNA sequence of MAB_1915 . MAB_1915 could therefore be associated with cell envelope permeability, and hence its role in intrinsic resistance to multiple drugs in M. abscessus , which presents it as a novel target for future development of effective antimicrobials to overcome intrinsic drug resistance in M. abscessus ., Importance: This study reports the role of a putative fadD (MAB_1915) in innate resistance to multiple drugs by M. abscessus , hence identifying MAB_1915 as a valuable target and providing a baseline for further mechanistic studies and development of effective antimicrobials to check the high level of intrinsic resistance in this pathogen., Competing Interests: The authors declare no conflict of interest.

Background: New and shorter regimens against multi-drug resistant tuberculosis (TB) remain urgently needed. To inform treatment duration in clinical trials, this study aimed to identify human pharmacokinetic equivalent doses, antimycobacterial and sterilizing activity of a novel regimen, containing bedaquiline, delamanid, moxifloxacin and sutezolid (BDMU), in the standard mouse model (BALB/c) of Mycobacterium tuberculosis (Mtb) infection., Methods: Treatment of mice with B25D0.6M200U200, B25D0.6M200, B25D0.6M200(U2003) or H10R10Z150E100 (isoniazid, rifampicin, pyrazinamide, ethambutol, HRZE), started 3 weeks after Mtb infection. Bactericidal activity was assessed after 1, 2, 3 and 4 months of treatment and relapse rates were assessed 3 months after completing treatment durations of 2, 3 and 4 months., Results: B25D0.6M200U200 generated human equivalent exposures in uninfected BALB/c mice. After 1 month of treatment, a higher bactericidal activity was observed for the B25D0.6M200U200 and the B25D0.6M200 regimen compared to the standard H10R10Z150E100 regimen. Furthermore, 3 months of therapy with both BDM-based regimens resulted in negative lung cultures, whereas all H10R10Z150E100 treated mice were still culture positive. After 3 months of therapy 7% and 13% of mice relapsed receiving B25D0.6M200U200 and B25D0.6M200, respectively, compared to 40% for H10R10Z150E100 treatment showing an increased sterilizing activity of both BDM-based regimens., Conclusions: BDM-based regimens, with and without sutezolid, have a higher efficacy than the HRZE regimen in the BALB/c model of TB, with some improvement by adding sutezolid. By translating these results to TB patients, this novel BDMU regimen should be able to reduce treatment duration by 25% compared to HRZE therapy., (© The Author(s) 2024. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy.)

Tuberculosis has posed a serious threat to human health. It is imperative to investigate the geographic prevalence of tuberculosis and medication resistance, as this information is essential for informing strategies for its prevention and treatment. Drug resistance was identified using a proportion method. Drug-resistant genes and pathways were predicted using whole genome sequencing. The drug resistance range of bedaquiline was identified using the microporous plate two-fold dilution method, and drug resistance genes were studied using sequencing. The study revealed that 19.99% of the tuberculosis cases had multidrug resistance. The genes of M. tuberculosis are predominantly involved in the synthesis of ABC transporters, two-component systems, and bacterial secretion systems, as well as in energy production and conversion, and lipid transport and metabolism. The genes encode for 82.45% of carbohydrate-related enzymes such as glycoside hydrolases, glycosyl transferases, and carbohydrate esterases. The minimum inhibitory concentration (MIC) of bedaquiline against clinical strains was approximately 0.06 μg/mL, with identified mutations in drug-resistant genes Rv0678, atpE, and pepQ, specifically V152A, P62A, and T222N, respectively. The multidrug resistance tuberculosis development was attributed to the strong medication resistance exhibited. It was concluded that tuberculosis had presented a high level of drug resistance. Phenotypic resistance was related to genes, existing potential genetic resistance in M. tuberculosis. Bedaquiline was found to possess effective antibacterial properties against M. tuberculosis., Competing Interests: The authors declare that there are no conflicts of interest regarding the publication of this paper., (Copyright: © 2024 Peng et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Liposomal formulations of antibiotics for inhalation offer the potential for the delivery of high drug doses, controlled drug release kinetics in the lung, and an excellent safety profile. In this study, we evaluated the in vivo performance of a liposomal formulation for the poorly soluble, antituberculosis agent, bedaquiline. Bedaquiline was encapsulated within monodisperse liposomes of ∼70 nm at a relatively high drug concentration (∼3.6 mg/mL). Formulations with or without fucose residues, which bind to C-type lectin receptors and mediate a preferential binding to macrophage mannose receptor, were prepared, and efficacy was assessed in an in vivo C3HeB/FeJ mouse model of tuberculosis infection (H37Rv strain). Seven intranasal instillations of 5 mg/kg bedaquiline formulations administered every second day resulted in a significant reduction in lung burden (∼0.4-0.6 Δlog 10 CFU), although no differences between fucosylated and nonfucosylated formulations were observed. A pharmacokinetic study in healthy, noninfected Balb/c mice demonstrated that intranasal administration of a single dose of 2.5 mg/kg bedaquiline liposomal formulation (fucosylated) improved the lung bioavailability 6-fold compared to intravenous administration of the same formulation at the same dose. Importantly, intranasal administration reduced systemic concentrations of the primary metabolite, N -desmethyl-bedaquiline (M2), compared with both intravenous and oral administration. This is a clinically relevant finding as the M2 metabolite is associated with a higher risk of QT-prolongation in predisposed patients. The results clearly demonstrate that a bedaquiline liposomal inhalation suspension may show enhanced antitubercular activity in the lung while reducing systemic side effects, thus meriting further nonclinical investigation.

The minimum bactericidal concentration (MBC) of antibiotics is an important parameter for the potency of a drug in eradicating a bacterium as well as an important measure of the potential of a drug candidate in research and development. We have established a fluorescence-based microscopy method for the determination of MBCs against the non-tuberculous mycobacterium Mycobacterium abscessus (Mycobacteroides abscessus) to simplify and accelerate the performance of MBC determination compared to counting colony forming units on agar. Bacteria are labelled with the trehalose-coupled dye 3HC-2-Tre and analysed in a 96-well plate. The results of the new method are consistent with MBC determination by plating on agar. The method was used to evaluate the bactericidality of the antibiotics rifabutin, moxifloxacin, amikacin, clarithromycin and bedaquiline. Bactericidal effects against M. abscessus were observed, which are consistent with literature data., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Adrian Richter reports financial support was provided by German Research Foundation. Adrian Richter reports financial support was provided by Deutsche Mukoviszidose Stiftung. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Background: Whole-genome sequencing (WGS)-based prediction of drug resistance in Mycobacterium tuberculosis has the potential to guide clinical decisions in the design of optimal treatment regimens., Methods: We utilized WGS to investigate drug resistance mutations in a 32-year-old Tanzanian male admitted to Kibong'oto Infectious Diseases Hospital with a history of interrupted multidrug-resistant tuberculosis treatment for more than three years. Before admission, he received various all-oral bedaquiline-based multidrug-resistant tuberculosis treatment regimens with unfavourable outcomes., Results: Drug susceptibility testing of serial M. tuberculosis isolates using Mycobacterium Growth Incubator Tubes culture and WGS revealed resistance to first-line anti-TB drugs, bedaquiline, and fluoroquinolones but susceptibility to linezolid, clofazimine, and delamanid. WGS of serial cultured isolates revealed that the Beijing (Lineage 2.2.2) strain was resistant to bedaquiline, with mutations in the mmpR5 gene (Rv0678. This study also revealed the emergence of two distinct subpopulations of bedaquiline-resistant tuberculosis strains with Asp47f and Glu49fs frameshift mutations in the mmpR5 gene, which might be the underlying cause of prolonged resistance. An individualized regimen comprising bedaquiline, delamanid, pyrazinamide, ethionamide, and para-aminosalicylic acid was designed. The patient was discharged home at month 8 and is currently in the ninth month of treatment. He reported no cough, chest pain, fever, or chest tightness but still experienced numbness in his lower limbs., Conclusion: We propose the incorporation of WGS in the diagnostic framework for the optimal management of patients with drug-resistant and extensively drug-resistant tuberculosis., (© 2024. The Author(s).)

Background: In 2018 the World Health Organization recommended a switch to an all oral bedaquiline-based second-line regimen for treatment of drug-resistant tuberculosis (DR-TB). How these new second-line regimens fare in comparison to first-line regimens for treatment of drug-sensitive tuberculosis (DS-TB) is not well known., Methods: In this study, we contemporaneously enrolled subjects with DS-TB (n = 31) or DR-TB (n = 23) and assessed their response to therapy with first-line (rifampin, isoniazid, ethambutol, pyrazinamide) or second-line (bedaquiline, pyrazinamide, levofloxacin, linezolid, clofazimine) regimens, respectively., Results: We found that the early bactericidal activity of first- and second-line regimens was similar during the first 2 weeks of therapy as determined by BACTEC MGIT, colony-forming units, and a liquid limiting dilution assay capable of detecting differentially detectable/culturable Mycobacterium tuberculosis. Furthermore, an identical percentage (77.8%) of subjects from the DS-TB and DR-TB cohorts converted to culture negative after 2 months of therapy., Conclusions: Despite presenting with more advanced disease at time of treatment, subjects with DR-TB receiving an all oral bedaquiline-based second-line treatment regimen displayed a similar microbiological response to therapy as subjects with DS-TB receiving a first-line treatment regimen., Competing Interests: Potential conflicts of interest. The authors declare no potential conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2023. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Background: The treatment of multidrug-resistant (MDR-) and extensively drug-resistant tuberculosis (XDR-TB) is a formidable challenge. Treatment of MDR- and XDR-TB using bedaquiline (BDQ) and delamanid (DLM), two newly introduced medications, is steadily increasing. This narrative review aimed to present a concise overview of the existing information regarding BDQ and DLM, and elucidate their antimicrobial characteristics, resistance mechanisms, synergism with other drugs, and side effects., Methods: To collect the required information about the antimicrobial properties, a search for scientific evidence from the Scopus, PubMed, and Embase databases was performed, and all recently published articles up to May 2024 were considered., Results: BDQ had potent antimicrobial effects on various types of nontuberculous mycobacteria (NTM), including rapid-growing and slow-growing species, and MDR/XDR Mycobacterium tuberculosis. The mechanisms of BDQ resistance in M. tuberculosis primarily involve mutations in three genes: atpE, mmpR (Rv0678) and pepQ. BDQ may have synergistic effects when combined with DLM, pyrazinamide, and pretomanid/linezolid. BDQ has a low incidence of side effects. The use of BDQ may prolong the QTc interval. Similarly, DLM showed potent antimicrobial effects on NTM and MDR/XDR M. tuberculosis. The main resistance mechanisms to DLM are induced by mutations in fbiA, fbiB, fbiC, fgd1, and ddn genes. The DLM had synergistic effects with BDQ and moxifloxacin. The DLM also has few side effects in some patients including QTc prolongation., Conclusion: BDQ and DLM are suitable antibiotics with few side effects for the treatment of MDR/XDR-TB. These antibiotics have synergistic effects when combined with other antituberculosis drugs., (© 2024 The Author(s). Journal of Clinical Laboratory Analysis published by Wiley Periodicals LLC.)

Background: The antibiotic bedaquiline is a key component of new WHO regimens for drug-resistant tuberculosis; however, predicting bedaquiline resistance from bacterial genotypes remains challenging. We aimed to understand the genetic mechanisms of bedaquiline resistance by analysing Mycobacterium tuberculosis isolates from South Africa., Methods: For this genomic analysis, we conducted whole-genome sequencing of Mycobacterium tuberculosis samples collected at two referral laboratories in Cape Town and Johannesburg, covering regions of South Africa with a high prevalence of tuberculosis. We used the tool ARIBA to measure the status of predefined genes that are associated with bedaquiline resistance. To produce a broad genetic landscape of M tuberculosis in South Africa, we extended our analysis to include all publicly available isolates from the European Nucleotide Archive, including isolates obtained by the CRyPTIC consortium, for which minimum inhibitory concentrations of bedaquiline were available., Findings: Between Jan 10, 2019, and July, 22, 2020, we sequenced 505 M tuberculosis isolates from 461 patients. Of the 64 isolates with mutations within the mmpR5 regulatory gene, we found 53 (83%) had independent acquisition of 31 different mutations, with a particular enrichment of truncated MmpR5 in bedaquiline-resistant isolates resulting from either frameshift mutations or the introduction of an insertion element. Truncation occurred across three M tuberculosis lineages, and were present in 66% of bedaquiline-resistant isolates. Although the distributions overlapped, the median minimum inhibitory concentration of bedaquiline was 0·25 mg/L (IQR 0·12-0·25) in mmpR5-disrupted isolates, compared with 0·06 mg/L (0·03-0·06) in wild-type M tuberculosis., Interpretation: Reduction in the susceptibility of M tuberculosis to bedaquiline has evolved repeatedly across the phylogeny. In our data, we see no evidence that this reduction has led to the spread of a successful strain in South Africa. Binary phenotyping based on the bedaquiline breakpoint might be inappropriate to monitor resistance to this drug. We recommend the use of minimum inhibitory concentrations in addition to MmpR5 truncation screening to identify moderate increases in resistance to bedaquiline., Funding: US Centers for Disease Control and Prevention., Competing Interests: Declaration of interests LWR reports funding from European Molecular Biology Laboratory’s European Bioinformatics Institute (EMBL-EBI). MRF reports grants from NIH/NIAID (5R01AI155765). All other authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Setting and Objective: To develop and evaluate newer molecular tests that identify drug resistance according to contemporary definitions in Tuberculous meningitis (TBM), the most severe form of EPTB., Design: 93 cerebrospinal fluid (CSF) specimens [41 culture-positive and 52 culture-negative], were subjected to Truenat MTB Plus assay along with chips for rifampicin, isoniazid, fluoroquinolones and bedaquiline resistance. The performance was compared against phenotypic drug susceptibility testing (pDST), Line probe assay (LPA) and gene sequencing., Results: Against pDST, Truenat chips had a sensitivity and specificity of 100%; 94.47%, 100%; 94.47%, 100%; 97.14% and 100%; 100%, respectively for rifampicin, isoniazid, fluoroquinolones and bedaquiline. Against LPA, all Truenat chips detected resistant isolates with 100% sensitivity; but 2 cases each of false-rifampicin and false-isoniazid resistance and 1 case of false-fluoroquinolone resistance was reported. Truenat drug chips gave indeterminate results in ∼25% cases, which were excluded. All cases reported indeterminate were found to be susceptible by pDST/LPA., Conclusion: The strategic drug resistance chips of Truenat Plus assay can contribute greatly to TB elimination by providing rapid and reliable detection of drug resistance pattern in TBM. Cases reported indeterminate require confirmation by other phenotypic and genotypic methods., Competing Interests: Declaration of competing interest The authors confirm that they have no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Bedaquiline (BDQ), a first-in-class diarylquinoline anti-tuberculosis drug, and its analogue, TBAJ-587, prevent the growth and proliferation of Mycobacterium tuberculosis by inhibiting ATP synthase 1,2 . However, BDQ also inhibits human ATP synthase 3 . At present, how these compounds interact with either M. tuberculosis ATP synthase or human ATP synthase is unclear. Here we present cryogenic electron microscopy structures of M. tuberculosis ATP synthase with and without BDQ and TBAJ-587 bound, and human ATP synthase bound to BDQ. The two inhibitors interact with subunit a and the c-ring at the leading site, c-only sites and lagging site in M. tuberculosis ATP synthase, showing that BDQ and TBAJ-587 have similar modes of action. The quinolinyl and dimethylamino units of the compounds make extensive contacts with the protein. The structure of human ATP synthase in complex with BDQ reveals that the BDQ-binding site is similar to that observed for the leading site in M. tuberculosis ATP synthase, and that the quinolinyl unit also interacts extensively with the human enzyme. This study will improve researchers' understanding of the similarities and differences between human ATP synthase and M. tuberculosis ATP synthase in terms of the mode of BDQ binding, and will allow the rational design of novel diarylquinolines as anti-tuberculosis drugs., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Objective: This study aims to estimate the overall in vitro activity of bedaquiline (BDQ) against clinical isolates of Mycobacterium abscessus complex (MABS) and M. avium complex (MAC), considering BDQ as a repurposed drug for non-tuberculous mycobacteria (NTM) infections., Methods: We conducted a systematic review of publications in PubMed/ MEDLINE, Web of Science, and Embase up to 15 April 2023. Studies were included if they followed the Clinical and Laboratory Standards Institute (CLSI) criteria for drug susceptibility testing (DST). Using a random effects model, we assessed the overall in vitro BDQ resistance rate in clinical isolates of MABS and MAC. Sources of heterogeneity were analysed using Cochran's Q and the I 2 statistic. All analyses were performed using CMA V3.0., Results: A total of 24 publications (19 reports for MABS and 11 for MAC) were included. Using 1 µg/mL and 2 µg/mL as the breakpoint for BDQ resistance, the pooled rates of in vitro BDQ resistance in clinical isolates of MABS were found to be 1.8% (95% confidence interval [CI], 0.7-4.6%) and 1.7% (95% CI, 0.6-4.4%), respectively. In the case of MAC, the pooled rates were 1.7% (95% CI, 0.4-6.9%) and 1.6% (95% CI, 0.4-6.8%) for 1 µg/mL and 2 µg/mL, respectively., Conclusion: This study reports the prevalence of BDQ resistance in clinical isolates of MABS and MAC. The findings suggest that BDQ holds potential as a repurposed drug for treating MABS and MAC infections., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Bedaquiline fumarate (BQF) is classified as a BCS class II drug and has poor water solubility and dissolution rate, which ultimately compromises bioavailability. The objective of this study is to improve the biopharmaceutical properties of BQF through a solid dispersion system by using Soluplus ® . Two solid dispersion systems were prepared i.e. binary solid dispersion (BSD) and ternary solid dispersion (TSD) where 14.31-fold and 20.43-fold increase in solubility of BQF was observed with BSD and TSD in comparison to BQF. In our previous research work, we explored the BSD and TSD of BQF with a crystalline polymer, poloxamer 188, which showed an increment in the solubility of BQF. In the current research, amorphous Soluplus ® polymer was selected to formulate BSD and TSD with BQF and showed higher solubility than poloxamer 188. The various solid and liquid state characterization results confirmed the presence of an amorphous form of BQF inside solid dispersion. The Fourier transform infrared spectroscopy showed no chemical interactions between BQF and polymer. The cellular uptake results demonstrated higher uptake in Caco-2 cell lines. Pharmacokinetic studies showed enhanced solubility and bioavailability of TSDs. Hence, the present research shows a promising formulation strategy for enhancing the biopharmaceutical performance of BQF by increasing its solubility.

Sputum culture reversion after conversion is an indicator of tuberculosis (TB) treatment failure. We analyze data from the endTB multi-country prospective observational cohort (NCT03259269) to estimate the frequency (primary endpoint) among individuals receiving a longer (18-to-20 month) regimen for multidrug- or rifampicin-resistant (MDR/RR) TB who experienced culture conversion. We also conduct Cox proportional hazard regression analyses to identify factors associated with reversion, including comorbidities, previous treatment, cavitary disease at conversion, low body mass index (BMI) at conversion, time to conversion, and number of likely-effective drugs. Of 1,286 patients, 54 (4.2%) experienced reversion, a median of 173 days (97-306) after conversion. Cavitary disease, BMI < 18.5, hepatitis C, prior treatment with second-line drugs, and longer time to initial culture conversion were positively associated with reversion. Reversion was uncommon. Those with cavitary disease, low BMI, hepatitis C, prior treatment with second-line drugs, and in whom culture conversion is delayed may benefit from close monitoring following conversion., (© 2024. The Author(s).)

A major challenge for tuberculosis (TB) drug development is to prioritize promising combination regimens from a large and growing number of possibilities. This includes demonstrating individual drug contributions to the activity of higher-order combinations. A BALB/c mouse TB infection model was used to evaluate the contributions of each drug and pairwise combination in the clinically relevant Nix-TB regimen [bedaquiline-pretomanid-linezolid (BPaL)] during the first 3 weeks of treatment at human equivalent doses. The rRNA synthesis (RS) ratio, an exploratory pharmacodynamic (PD) marker of ongoing Mycobacterium tuberculosis rRNA synthesis, together with solid culture CFU counts and liquid culture time to positivity (TTP) were used as PD markers of treatment response in lung tissue; and their time-course profiles were mathematically modeled using rate equations with pharmacologically interpretable parameters. Antimicrobial interactions were quantified using Bliss independence and Isserlis formulas. Subadditive (or antagonistic) and additive effects on bacillary load, assessed by CFU and TTP, were found for bedaquiline-pretomanid and linezolid-containing pairs, respectively. In contrast, subadditive and additive effects on rRNA synthesis were found for pretomanid-linezolid and bedaquiline-containing pairs, respectively. Additionally, accurate predictions of the response to BPaL for all three PD markers were made using only the single-drug and pairwise effects together with an assumption of negligible three-way drug interactions. The results represent an experimental and PD modeling approach aimed at reducing combinatorial complexity and improving the cost-effectiveness of in vivo systems for preclinical TB regimen development., Competing Interests: N.D.W. and G.T.R. are listed as co-inventors on US patent no. 16/632,310 which pertains to the RS ratio.

Background: Tuberculosis (TB) continues to pose a threat to communities worldwide and remains a significant public health issue in several countries. We assessed the role of heteroresistance and efflux pumps in bedaquiline (BDQ)-resistant Mycobacterium tuberculosis isolates., Methods: Nineteen clinical isolates were included in the study, of which fifteen isolates were classified as MDR or XDR, while four isolates were fully susceptible. To evaluate BDQ heteroresistance, the Microplate Alamar Blue Assay (MABA) method was employed. For screening mixed infections, MIRU-VNTR was performed on clinical isolates. Mutations in the atpE and Rv0678 genes were determined based on next-generation sequencing data. Additionally, real-time PCR was applied to assess the expression of efflux pump genes in the absence and presence of verapamil (VP)., Results: All 15 drug-resistant isolates displayed resistance to BDQ. Among the 19 total isolates, 21.05% (4/19) exhibited a heteroresistance pattern to BDQ. None of the isolates carried a mutation of the atpE and Rv0678 genes associated with BDQ resistance. Regarding the MIRU-VNTR analysis, most isolates (94.73%) showed the Beijing genotype. Fifteen (78.9%) isolates showed a significant reduction in BDQ MIC after VP treatment. The efflux pump genes of Rv0676c, Rv1258c, Rv1410c, Rv1634, Rv1819, Rv2459, Rv2846, and Rv3065 were overexpressed in the presence of BDQ., Conclusions: Our results clearly demonstrated the crucial role of heteroresistance and efflux pumps in BDQ resistance. Additionally, we established a direct link between the Rv0676c gene and BDQ resistance. The inclusion of VP significantly reduced the MIC of BDQ in both drug-susceptible and drug-resistant clinical isolates., (© 2024. The Author(s).)

Multidrug-resistant tuberculosis (MDR-TB) patients not cured at the time of stopping treatment are exposed to Minimum Inhibitory Concentration (MIC) and sub-MIC levels for many months after discontinuing bedaquiline (BDQ) or clofazimine (CFZ) treatment. In vitro cultures treated with BDQ and CFZ sub-MIC concentrations clearly showed enrichment in the Rv0678 mutant population, demonstrating that pre-existing Rv0678 mutants can be selected by sub-MIC concentrations of BDQ and CFZ if not protected by an alternative MDR-TB treatment., Competing Interests: All authors were Janssen employees at the time of performing this work. Cristina Villellas and Koen Andries have J&J stock.

The combination of bedaquiline, pretomanid, and linezolid (BPaL) has become a preferred regimen for treating multidrug- and extensively drug-resistant tuberculosis (TB). However, treatment-limiting toxicities of linezolid and reports of emerging bedaquiline and pretomanid resistance necessitate efforts to develop new short-course oral regimens. We recently found that the addition of GSK2556286 increases the bactericidal and sterilizing activity of BPa-containing regimens in a well-established BALB/c mouse model of tuberculosis. Here, we used this model to evaluate the potential of new regimens combining bedaquiline or the more potent diarylquinoline TBAJ-587 with GSK2556286 and the DprE1 inhibitor TBA-7371, all of which are currently in early-phase clinical trials. We found the combination of bedaquiline, GSK2556286, and TBA-7371 to be more active than the first-line regimen and nearly as effective as BPaL in terms of bactericidal and sterilizing activity. In addition, we found that GSK2556286 and TBA-7371 were as effective as pretomanid and the novel oxazolidinone TBI-223 when either drug pair was combined with TBAJ-587 and that the addition of GSK2556286 increased the bactericidal activity of the TBAJ-587, pretomanid, and TBI-223 combination. We conclude that GSK2556286 and TBA-7371 have the potential to replace pretomanid, an oxazolidinone, or both components, in combination with bedaquiline or TBAJ-587., Competing Interests: David Barros-Aguirre and Elena Jimenez-Navarro are employees of GlaxoSmithKline. Eric L. Nuermberger receives research support from Janssen and the TB Alliance. Lluis Ballell was previously an employee of GlaxoSmithKline and is currently an employee of Janssen.

In the accompanying study, Nimmo and colleagues estimated the dates of emergence of mutations in mmpR5 (Rv0678), the most important resistance gene to the anti-tuberculosis drug bedaquiline, in over 3500 geographically diverse Mycobacterium tuberculosis genomes. This provided important insights to improve the design and analysis of clinical trials, as well as the World Health Organization catalogue of resistance mutations, the global reference for interpreting genotypic antimicrobial susceptibility testing results., (© 2024. The Author(s).)

Bedaquiline is currently a key drug for treating multidrug-resistant or rifampin-resistant tuberculosis. We report and discuss the unusual development of resistance to bedaquiline in a teenager in Namibia, despite an optimal background regimen and adherence. The report highlights the risk for bedaquiline resistance development and the need for rapid drug-resistance testing.

Background: Emerging resistance to bedaquiline (BDQ) threatens to undermine advances in the treatment of drug-resistant tuberculosis (DRTB). Characterizing serial Mycobacterium tuberculosis (Mtb) isolates collected during BDQ-based treatment can provide insights into the etiologies of BDQ resistance in this important group of DRTB patients., Methods: We measured mycobacteria growth indicator tube (MGIT)-based BDQ minimum inhibitory concentrations (MICs) of Mtb isolates collected from 195 individuals with no prior BDQ exposure who were receiving BDQ-based treatment for DRTB. We conducted whole-genome sequencing on serial Mtb isolates from all participants who had any isolate with a BDQ MIC >1 collected before or after starting treatment (95 total Mtb isolates from 24 participants)., Results: Sixteen of 24 participants had BDQ-resistant TB (MGIT MIC ≥4 µg/mL) and 8 had BDQ-intermediate infections (MGIT MIC = 2 µg/mL). Participants with pre-existing resistance outnumbered those with resistance acquired during treatment, and 8 of 24 participants had polyclonal infections. BDQ resistance was observed across multiple Mtb strain types and involved a diverse catalog of mmpR5 (Rv0678) mutations, but no mutations in atpE or pepQ. Nine pairs of participants shared genetically similar isolates separated by <5 single nucleotide polymorphisms, concerning for potential transmitted BDQ resistance., Conclusions: BDQ-resistant TB can arise via multiple, overlapping processes, including transmission of strains with pre-existing resistance. Capturing the within-host diversity of these infections could potentially improve clinical diagnosis, population-level surveillance, and molecular diagnostic test development., Competing Interests: Potential conflicts of interest. N. S. S. reports the following grants or contracts unrelated to this work: NIH/NIAID P30AI168386 and NIH/NIAID K24AI165099. T. S. B. reports reimbursement for travel and accommodation expenses for an invited presentation for the RePORT TB International Annual Meeting 2022. G. Meintjes reports grants or contracts from the Wellcome Trust, National Institutes of Health, ImmunityBio, UK National Institute for Health and Care Research, European and Developing Countries Clinical Trials Partnership, and South African Medical Research Council; consulting fees from Aid for AIDS; payment or honoraria for speaking or educational events from Gilead Sciences; and participation on Data Safety Monitoring or Advisory Boards for Otsuka, Drugs for Neglected Diseases Initiative, National Institutes of Health, and the Bill and Melinda Gates Medical Research Institute. All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2023. Published by Oxford University Press on behalf of Infectious Diseases Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The F 1 F O -ATP synthase engine is essential for viability and growth of nontuberculous mycobacteria (NTM) by providing the biological energy ATP and keeping ATP homeostasis under hypoxic stress conditions. Here, we report the discovery of the diarylquinoline TBAJ-5307 as a broad spectrum anti-NTM inhibitor, targeting the F O domain of the engine and preventing rotation and proton translocation. TBAJ-5307 is active at low nanomolar concentrations against fast- and slow-growing NTM as well as clinical isolates by depleting intrabacterial ATP. As demonstrated for the fast grower Mycobacterium abscessus, the compound is potent in vitro and in vivo, without inducing toxicity. Combining TBAJ-5307 with anti-NTM antibiotics or the oral tebipenem-avibactam pair showed attractive potentiation. Furthermore, the TBAJ-5307-tebipenem-avibactam cocktail kills the pathogen, suggesting a novel oral combination for the treatment of NTM lung infections., Competing Interests: Conflict of interest G. G., R. W. B., P. R., P. S.-L., and L. K. are inventors on the patent 23306555.6, which is related to the inhibitor described in this article. All other authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Non-tuberculous mycobacteria (NTM) infections are increasing in incidence and associated mortality. NTM are naturally resistant to a variety of antibiotics, complicating treatment. We conducted a literature assessment on the efficacy of bedaquiline in treating NTM species in vitro and in vivo (animal models and humans); meta-analyses were performed where possible., Method: Four databases were searched using specific terms. Publications were included according to predefined criteria. Bedaquiline's impact on NTM in vitro, MICs and epidemiological cut-off (ECOFF) values were evaluated. A meta-analysis of bedaquiline efficacy against NTM infections in animal models was performed. Culture conversion, cure and/or relapse-free cure were used to evaluate the efficacy of bedaquiline in treating NTM infection in humans., Results: Fifty studies met the inclusion criteria: 33 assessed bedaquiline's impact on NTM in vitro, 9 in animal models and 8 in humans. Three studies assessed bedaquiline's efficacy both in vitro and in vivo. Due to data paucity, an ECOFF value of 0.5 mg/mL was estimated for Mycobacterium abscessus only. Meta-analysis of animal studies showed a 1.86× reduction in bacterial load in bedaquiline-treated versus no treatment within 30 days. In humans, bedaquiline-including regimens were effective in treating NTM extrapulmonary infection but not pulmonary infection., Conclusions: Bedaquiline demonstrated strong antibacterial activity against various NTM species and is a promising drug to treat NTM infections. However, data on the genomic mutations associated with bedaquiline resistance were scarce, preventing statistical analyses for most mutations and NTM species. Further studies are urgently needed to better inform treatment strategies., (© The Author(s) 2023. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy.)

The emergence of drug resistant Mycobacterium tuberculosis strains increases the burden on the treatment of tuberculosis. In this study, through in-silico transcriptome analysis of drug-treated M. tuberculosis samples, novel drug targets for the treatment of drug resistance in tuberculosis were identified. Gene expression datasets of tuberculosis patients samples treated with different antibiotics (Isoniazid, Rifampicin, Pyrazinamide, Bedaquiline and Linezolid) were considered in this study. DESeq2 was used to identify the differentially regulated genes. Novel genes which were up-regulated during antibiotic treatment were identified which could be antibiotic resistance factors. Further, to understand the resistance mechanism of the novel genes, we performed gene ontology and gene network analysis for the differentially up-regulated genes. Thus, the in-silico transcriptome analysis paves way for a deeper understanding of the antibiotic resistance in M. tuberculosis., Competing Interests: Conflicts of interest The authors has none to declare, (Copyright © 2023 Tuberculosis Association of India. Published by Elsevier B.V. All rights reserved.)

Based on the assessment of new evidence, the World Health Organization (WHO) updated its guidelines for the treatment of drug-resistant tuberculosis (TB) in December 2022. The new recommendations and the latest study data made it necessary to update the existing guideline on the treatment of at least rifampicin-resistant TB (RR-TB) for the German-speaking countries, replacing the respective chapters of the treatment guidelines published in 2022. A shortened treatment of proven RR-TB and multidrug-resistant TB for at least 6 months using the fixed and non-modifiable drug combination of bedaquiline, pretomanid, linezolid, and moxifloxacin (BPaLM) is now also recommended for Austria, Germany, and Switzerland under certain conditions considering the existing barriers for the implementation of the new treatment regimen. For the treatment of pre-extensively drug-resistant (pre-XDR-) TB, an individualized treatment for 18 months continues to be the primary recommendation. The non-modifiable drug combination of bedaquiline, pretomanid, and linezolid (BPaL) may be used alternatively in selected pre-XDR-TB cases, provided that all prerequisites are met. The necessary requirements for using BPaLM and BPaL are presented in detail in this amendment to the consensus-based TB treatment guideline for adult patients., (© 2024 The Author(s). Published by S. Karger AG, Basel.)

The anti-tuberculosis therapeutic bedaquiline (BDQ) is used against Mycobacterium abscessus. In M. abscessus BDQ is only bacteriostatic and less potent compared to M. tuberculosis or M. smegmatis. Here we demonstrate its reduced ATP synthesis inhibition against M. abscessus inside-out vesicles, including the F 1 F O -ATP synthase. Molecular dynamics simulations and binding free energy calculations highlight the differences in drug-binding of the M. abscessus and M. smegmatis F O -domain at the lagging site, where the drug deploys its mechanistic action, inhibiting ATP synthesis. These data pave the way for improved anti-M. abscessus BDQ analogs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Toxoplasma gondii is a zoonotic parasite that infects one-third of the world's population and nearly all warm-blooded animals. Due to the complexity of T. gondii's life cycle, available treatment options have limited efficacy. Thus, there is an urgent need to develop new compounds or repurpose existing drugs with potent anti-Toxoplasma activity. This study demonstrates that bedaquiline (BDQ), an FDA-approved diarylquinoline antimycobacterial drug for the treatment of tuberculosis, potently inhibits the tachyzoites of T. gondii. At a safe concentration, BDQ displayed a dose-dependent inhibition on T. gondii growth with a half-maximal effective concentration (EC 50 ) of 4.95 μM. Treatment with BDQ significantly suppressed the proliferation of T. gondii tachyzoites in the host cell, while the invasion ability of the parasite was not affected. BDQ incubation shrunk the mitochondrial structure and decreased the mitochondrial membrane potential and ATP level of T. gondii parasites. In addition, BDQ induced elevated ROS and led to autophagy in the parasite. By transcriptomic analysis, we found that oxidative phosphorylation pathway genes were significantly disturbed by BDQ-treated parasites. More importantly, BDQ significantly reduces brain cysts for the chronically infected mice. These results suggest that BDQ has potent anti-T. gondii activity and may impair its mitochondrial function by affecting proton transport. This study provides bedaquiline as a potential alternative drug for the treatment of toxoplasmosis, and our findings may facilitate the development of new effective drugs for the treatment of toxoplasmosis., (© 2023. The Author(s).)

Contezolid is a new oxazolidinone with in vitro and in vivo activity against Mycobacterium tuberculosis comparable to that of linezolid. Pre-clinical and clinical safety studies suggest it may be less toxic than linezolid, making contezolid a potential candidate to replace linezolid in the treatment of drug-resistant tuberculosis. We evaluated the dose-ranging activity of contezolid, alone and in combination with bedaquiline and pretomanid, and compared it with linezolid at similar doses, in an established BALB/c mouse model of tuberculosis. Contezolid had an MIC of 1 µg/mL, similar to linezolid, and exhibited similar bactericidal activity in mice. Contezolid-resistant mutants selected in vitro had 32- to 64-fold increases in contezolid MIC and harbored mutations in the mce3R gene. These mutants did not display cross-resistance to linezolid. Our results indicate that contezolid has the potential to replace linezolid in regimens containing bedaquiline and pretomanid and likely other regimens., Competing Interests: Barry Hafkin is an employee of MicuRx Pharmaceuticals, Khisimuzi Mdluli is an employee of the Bill & Melinda Gates Medical Research Institute, and Nader Fotouhi is an employee of the Global Alliance for Tuberculosis Drug Development.

Background: Bedaquiline (BDQ), by targeting the electron transport chain and having a long half-life, is a good candidate to simplify leprosy treatment. Our objectives were to (i) determine the minimal effective dose (MED) of BDQ administered orally, (ii) evaluate the benefit of combining two inhibitors of the respiratory chain, BDQ administered orally and clofazimine (CFZ)) and (iii) evaluate the benefit of an intramuscular injectable long-acting formulation of BDQ (intramuscular BDQ, BDQ-LA IM), in a murine model of leprosy., Methodology/principal Findings: To determine the MED of BDQ administered orally and the benefit of adding CFZ, 100 four-week-old female nude mice were inoculated in the footpads with 5x103 bacilli of M. leprae strain THAI53. Mice were randomly allocated into: 1 untreated group, 5 groups treated with BDQ administered orally (0.10 to 25 mg/kg), 3 groups treated with CFZ 20 mg/kg alone or combined with BDQ administered orally 0.10 or 0.33 mg/kg, and 1 group treated with rifampicin (RIF) 10 mg/kg. Mice were treated 5 days a week during 24 weeks. To evaluate the benefit of the BDQ-LA IM, 340 four-week-old female swiss mice were inoculated in the footpads with 5x103 to 5x101 bacilli (or 5x100 for the untreated control group) of M. leprae strain THAI53. Mice were randomly allocated into the following 11 groups treated with a single dose (SD) or 3 doses (3D) 24h after the inoculation: 1 untreated group, 2 treated with RIF 10 mg/kg SD or 3D, 8 treated with BDQ administered orally or BDQ-LA IM 2 or 20 mg/kg, SD or 3D. Twelve months later, mice were sacrificed and M. leprae bacilli enumerated in the footpad. All the footpads became negative with BDQ at 3.3 mg/kg. The MED of BDQ administered orally against M. leprae in this model is therefore 3.3 mg/kg. The combination of CFZ and BDQ 10-fold lower than this MED did not significantly increase the bactericidal activity of CFZ. The BDQ-LA IM displayed similar or lower bactericidal activity than the BDQ administered orally., Conclusion: We demonstrated that the MED of BDQ administered orally against M. leprae was 3.3 mg/kg in mice and BDQ did not add significantly to the efficacy of CFZ at the doses tested. BDQ-LA IM was similar or less active than BDQ administered orally at equivalent dosing and frequency but should be tested at higher dosing in order to reach equivalent exposure in further experiments., Competing Interests: We have read the journal’s policy and the authors of this manuscript have the following competing interests: NL is an employee of Janssen and KA has retired from Janssen; they participated to the methodology design and writing of the manuscript. AA, AC, VJ, NV have no conflict of interest to declare except funding aforementioned., (Copyright: © 2023 Chauffour et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

ESX-3 is a secretion pathway which is essential for mycobactin-mediated iron acquisition under iron-limited conditions. Although present in all Mycobacterium sp., ESX-3 remains to be elucidated in Mycobacterium abscessus. In the study reported here, impaired ESX-3 seriously restricts the growth of M. abscesses under iron-limited conditions; growth is salvaged by functional ESX-3 or iron supplementation. Notably, impaired ESX-3 does not kill M. abscesses when environmental iron is insufficient but induces persistence to bedaquiline, a diarylquinoline class antibiotic used to treat multidrug-resistant mycobacteria. One potential mechanism contributing to persistence is the iron deficiency due to impaired ESX-3 suppressing succinate dehydrogenase activity, which dysregulates the tricarboxylic acid cycle and inactivates bedaquiline. Experiments conducted here also demonstrate that the regulator, MtrA, can bind ESX-3 and promote the survival of M. abscessus. As such, this study suggests that a novel pathway involving MtrA, ESX-3, iron metabolism, and the TCA cycle contributes to bedaquiline persistence in M. abscesses growing under iron-limited conditions., (© 2023 Wiley-VCH GmbH.)

Mycobacteria, such as Mycobacterium tuberculosis, depend on the activity of adenosine triphosphate (ATP) synthase for growth. The diarylquinoline bedaquiline (BDQ), a mycobacterial ATP synthase inhibitor, is an important medication for treatment of drug-resistant tuberculosis but suffers from off-target effects and is susceptible to resistance mutations. Consequently, both new and improved mycobacterial ATP synthase inhibitors are needed. We used electron cryomicroscopy and biochemical assays to study the interaction of Mycobacterium smegmatis ATP synthase with the second generation diarylquinoline TBAJ-876 and the squaramide inhibitor SQ31f. The aryl groups of TBAJ-876 improve binding compared with BDQ, while SQ31f, which blocks ATP synthesis ~10 times more potently than ATP hydrolysis, binds a previously unknown site in the enzyme's proton-conducting channel. Remarkably, BDQ, TBAJ-876, and SQ31f all induce similar conformational changes in ATP synthase, suggesting that the resulting conformation is particularly suited for drug binding. Further, high concentrations of the diarylquinolines uncouple the transmembrane proton motive force while for SQ31f they do not, which may explain why high concentrations of diarylquinolines, but not SQ31f, have been reported to kill mycobacteria., (© 2023 The Authors.)

The diarylquinoline bedaquiline (BDQ) is an FDA-approved drug for the treatment of multidrug-resistant tuberculosis that targets the mycobacterial adenosine triphosphate (ATP) synthase, a key enzyme in cellular respiration. In a recent study, Courbon et al (2023) examine the interaction between Mycobacterium smegmatis ATP synthase with the second generation diarylquinoline TBAJ-876 and the squaramide inhibitor SQ31f, showing that both drugs prevent the rotatory motions needed for enzymatic function., (© 2023 The Authors.)

Bedaquiline (BDQ) is an effective drug for the treatment of drug-resistant tuberculosis. Mutations in atpE , which encodes the target of BDQ, are associated with large increases in MICs. Mutations in Rv0678 that derepress the transcription of the MmpL5-MmpS5 efflux transporter are associated with smaller increases in MICs. However, Rv0678 mutations are the most common mutations that are associated with BDQ resistance in clinical isolates, and they also confer cross-resistance to clofazimine (CFZ). To investigate the mechanism of BDQ resistance and the correlation between Rv0678 mutations and target-based atpE mutations, M. tuberculosis strains were exposed to different concentrations of BDQ or CFZ to select Rv0678 mutations and atpE mutations. Gene overexpression strains were constructed to illustrate the roles of MmpL5 and MmpS5. A quantitative proteome analysis was performed to compare the BDQ-resistant mutants to the isogenic strain H37Rv. Here, we report that the Rv0678 mutations were more readily selected than were the atpE mutations at low concentrations of BDQ or CFZ. The atpE mutations were selected by high concentrations of BDQ exposure. The overexpression of both mmpL5 and mmpS5 reduced the susceptibility of Mycobacterium tuberculosis to BDQ and CFZ. Secreted immunogenic proteins and proteins involved in the biosynthesis and transport of phthiocerol dimycocerosates were associated with Rv0678 mutations conferring BDQ resistance in the proteome analysis. In conclusion, exposure to different bedaquiline concentrations resulted in the selection of different mutations. The coexpression of MmpL5 and MmpS5 contributed to drug resistance and upregulated pathogenic proteins in M. tuberculosis, suggesting MmpL5-MmpS5 as a new potential target for antituberculosis drug development. These results warrant further surveillance for the evolution of BDQ resistance during clinical usage.

Drug-resistant tuberculosis is a serious global health threat. Bedaquiline (BDQ) is a relatively new core drug, targeting the respiratory chain in Mycobacterium tuberculosis (Mtb). While mutations in the BDQ target gene, atpE, are rare in clinical isolates, mutations in the Rv0678 gene, a transcriptional repressor regulating the efflux pump MmpS5-MmpL5, are increasingly observed, and have been linked to worse treatment outcomes. Nevertheless, underlying mechanisms of (cross)-resistance remain incompletely resolved. Our study aims to distinguish resistance associated variants from other polymorphisms, by assessing the in vitro onset of mutations under drug pressure, combined with their impact on minimum inhibitory concentrations (MICs) and on protein stability. For this purpose, isolates were exposed in vitro to sub-lethal concentrations of BDQ or clofazimine (CFZ). Selected colonies had BDQ- and CFZ-MICs determined on 7H10 and 7H11 agar. Sanger sequencing and additional Deeplex Myc-TB and whole genome sequencing (WGS) for a subset of isolates were used to search for mutations in Rv0678, atpE and pepQ. In silico characterization of relevant mutations was performed using computational tools. We found that colonies that grew on BDQ medium had mutations in Rv0678, atpE or pepQ, while CFZ-exposed isolates presented mutations in Rv0678 and pepQ, but none in atpE. Twenty-eight Rv0678 mutations had previously been described among in vitro selected mutants or in patients' isolates, while 85 were new. Mutations were scattered across the Rv0678 gene without apparent hotspot. While most Rv0678 mutations led to an increased BDQ- and/or CFZ-MIC, only a part of them surpassed the critical concentration (69.1% for BDQ and 87.9% for CFZ). Among the mutations leading to elevated MICs for BDQ and CFZ, we report a synonymous Val1Val mutation in the Rv0678 start codon. Finally, in silico characterization of Rv0678 mutations suggests that especially the C46R mutant may render Rv0678 less stable., (© 2023. The Author(s).)

Molecular detection of bedaquiline resistant tuberculosis is challenging as only a small proportion of mutations in candidate bedaquiline resistance genes have been statistically associated with phenotypic resistance. We introduced two mutations, atpE Ile66Val and Rv0678 Thr33Ala, in the Mycobacterium tuberculosis H37Rv reference strain using homologous recombineering or recombination to investigate the phenotypic effect of these mutations. The genotype of the resulting strains was confirmed by Sanger- and whole genome sequencing, and bedaquiline susceptibility was assessed by minimal inhibitory concentration (MIC) assays. The impact of the mutations on protein stability and interactions was predicted using mutation Cutoff Scanning Matrix (mCSM) tools. The atpE Ile66Val mutation did not elevate the MIC above the critical concentration (MIC 0.25-0.5 µg/ml), while the MIC of the Rv0678 Thr33Ala mutant strains (> 1.0 µg/ml) classifies the strain as resistant, confirming clinical findings. In silico analyses confirmed that the atpE Ile66Val mutation minimally disrupts the bedaquiline-ATP synthase interaction, while the Rv0678 Thr33Ala mutation substantially affects the DNA binding affinity of the MmpR transcriptional repressor. Based on a combination of wet-lab and computational methods, our results suggest that the Rv0678 Thr33Ala mutation confers resistance to BDQ, while the atpE Ile66Val mutation does not, but definite proof can only be provided by complementation studies given the presence of secondary mutations., (© 2023. The Author(s).)

Objectives: Bedaquiline (BDQ) is a potent drug for treating drug-resistant tuberculosis (TB). Here, we analysed the resistance profiles of BDQ in CFZ-resistant clinical isolates and investigated the clinical risk factors of BDQ and CFZ cross/co-resistance., Methods: The AlarmarBlue microplate assay was performed to determine the minimum inhibitory concentration (MIC) of the CFZ-resistant Mycobacterium tuberculosis (MTB) clinical isolates to CFZ and BDQ. The clinical characteristics of the respective patients were analysed to explore the possible risk factors of BDQ resistance. The drug-resistance-associated genes including Rv0678, Rv1979c, atpE, pepQ and Rv1453 were sequenced and analysed., Results: A total of 72 clinical CFZ-resistant MTB isolates were collected; among these, half were identified as BDQ-resistant. The MIC value of BDQ closely correlated with CFZ (Spearman's q = 0.766, P < 0.005). Among the isolates with a MIC of CFZ ≥4 mg/L, 92.31% (12/13) were resistant to BDQ. Pre-XDR and exposure to BDQ or CFZ are the major risk factors for concurrent BDQ resistance. Among the 36 cross/co-resistant isolates, 50% (18/36) had mutations in Rv0678, 8.3% (3/36) had mutations in Rv0678+Rv1453, 5.6% (2/36) had mutations in Rv0678+Rv1979c, 2.8% (1/36) had mutations in Rv0678+Rv1979c+Rv1453, 2.8% (1/36) had mutations in atpE+Rv0678+Rv1453, 2.8% (1/36) had mutations in Rv1979c, and 27.7% (10/36) had no variations in the target genes., Conclusion: Nearly half of the CFZ-resistant isolates were still sensitive to BDQ, whereas this rate dramatically decreased among patients with pre-XDR TB or those who had been exposed to BDQ or CFZ., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

New classes of antitubercular drugs, diarylquinolines and nitroimidazoles, have been associated with improved outcomes in the treatment of drug-resistant tuberculosis, but that success is threatened by emerging drug resistance. We report a case of bedaquiline and delamanid resistance in a 55-year-old woman in South Africa with extensively drug-resistant tuberculosis and known HIV.

Background: Bedaquiline is a core drug for the treatment of multidrug-resistant tuberculosis; however, the understanding of resistance mechanisms is poor, which is hampering rapid molecular diagnostics. Some bedaquiline-resistant mutants are also cross-resistant to clofazimine. To decipher bedaquiline and clofazimine resistance determinants, we combined experimental evolution, protein modelling, genome sequencing, and phenotypic data., Methods: For this in-vitro and in-silico data analysis, we used a novel in-vitro evolutionary model using subinhibitory drug concentrations to select bedaquiline-resistant and clofazimine-resistant mutants. We determined bedaquiline and clofazimine minimum inhibitory concentrations and did Illumina and PacBio sequencing to characterise selected mutants and establish a mutation catalogue. This catalogue also includes phenotypic and genotypic data of a global collection of more than 14 000 clinical Mycobacterium tuberculosis complex isolates, and publicly available data. We investigated variants implicated in bedaquiline resistance by protein modelling and dynamic simulations., Findings: We discerned 265 genomic variants implicated in bedaquiline resistance, with 250 (94%) variants affecting the transcriptional repressor (Rv0678) of the MmpS5-MmpL5 efflux system. We identified 40 new variants in vitro, and a new bedaquiline resistance mechanism caused by a large-scale genomic rearrangement. Additionally, we identified in vitro 15 (7%) of 208 mutations found in clinical bedaquiline-resistant isolates. From our in-vitro work, we detected 14 (16%) of 88 mutations so far identified as being associated with clofazimine resistance and also seen in clinically resistant strains, and catalogued 35 new mutations. Structural modelling of Rv0678 showed four major mechanisms of bedaquiline resistance: impaired DNA binding, reduction in protein stability, disruption of protein dimerisation, and alteration in affinity for its fatty acid ligand., Interpretation: Our findings advance the understanding of drug resistance mechanisms in M tuberculosis complex strains. We have established an extended mutation catalogue, comprising variants implicated in resistance and susceptibility to bedaquiline and clofazimine. Our data emphasise that genotypic testing can delineate clinical isolates with borderline phenotypes, which is essential for the design of effective treatments., Funding: Leibniz ScienceCampus Evolutionary Medicine of the Lung, Deutsche Forschungsgemeinschaft, Research Training Group 2501 TransEvo, Rhodes Trust, Stanford University Medical Scientist Training Program, National Institute for Health and Care Research Oxford Biomedical Research Centre, Oxford University Hospitals NHS Foundation Trust, Bill & Melinda Gates Foundation, Wellcome Trust, and Marie Skłodowska-Curie Actions., Competing Interests: Declaration of interests AS is the cofounder and owner of shares in BiKi Technology, which sells the BiKi Life Sciences software suite used in this study's analysis. PWF has received consulting fees from Global Pathogen Analysis Service. All other authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 license. Published by Elsevier Ltd.. All rights reserved.)

In Mycobacterium tuberculosis, bedaquiline and clofazimine resistance occurs primarily through Rv0678 variants, a gene encoding a repressor protein that regulates mmpS5 / mmpL5 efflux pump gene expression. Despite the shared effect of both drugs on efflux, little else is known about other pathways affected. We hypothesized that in vitro generation of bedaquiline- or clofazimine-resistant mutants could provide insight into additional mechanisms of action. We performed whole-genome sequencing and determined phenotypic MICs for both drugs on progenitor and mutant progenies. Mutants were induced through serial passage on increasing concentrations of bedaquiline or clofazimine. Rv0678 variants were identified in both clofazimine- and bedaquiline-resistant mutants, with concurrent atpE SNPs occurring in the latter. Of concern was the acquisition of variants in the F420 biosynthesis pathway in clofazimine-resistant mutants obtained from either a fully susceptible ( fbiD : del555GCT) or rifampicin mono-resistant ( fbiA : 283delTG and T862C) progenitor. The acquisition of these variants possibly implicates a shared pathway between clofazimine and nitroimidazoles. Pathways associated with drug tolerance and persistence, F420 biosynthesis, glycerol uptake and metabolism, efflux, and NADH homeostasis appear to be affected following exposure to these drugs. Shared genes affected by both drugs include Rv0678 , glpK , nuoG , and uvrD1 . Genes with variants in the bedaquiline resistant mutants included atpE , fadE28 , truA , mmpL5 , glnH , and pks8 , while clofazimine-resistant mutants displayed ppsD , fbiA , fbiD , mutT3 , fadE18 , Rv0988 , and Rv2082 variants. These results show the importance of epistatic mechanisms as a means of responding to drug pressure and highlight the complexity of resistance acquisition in M. tuberculosis.

A regimen comprised of bedaquiline (BDQ, or B), pretomanid, and linezolid (BPaL) is the first oral 6-month regimen approved by the U.S. Food and Drug Administration and recommended by the World Health Organization for the treatment of extensively drug-resistant tuberculosis. We used a well-established BALB/c mouse model of tuberculosis to evaluate the treatment-shortening potential of replacing bedaquiline with either of two new, more potent diarylquinolines, TBAJ-587 and TBAJ-876, in early clinical trials. We also evaluated the effect of replacing linezolid with a new oxazolidinone, TBI-223, exhibiting a larger safety margin with respect to mitochondrial toxicity in preclinical studies. Replacing bedaquiline with TBAJ-587 at the same 25-mg/kg dose significantly reduced the proportion of mice relapsing after 2 months of treatment, while replacing linezolid with TBI-223 at the same 100-mg/kg dose did not significantly change the proportion of mice relapsing. Replacing linezolid or TBI-223 with sutezolid in combination with TBAJ-587 and pretomanid significantly reduced the proportion of mice relapsing. In combination with pretomanid and TBI-223, TBAJ-876 at 6.25 mg/kg was equipotent to TBAJ-587 at 25 mg/kg. We conclude that replacement of bedaquiline with these more efficacious and potentially safer diarylquinolines and replacement of linezolid with potentially safer and at least as efficacious oxazolidinones in the clinically successful BPaL regimen may lead to superior regimens capable of treating both drug-susceptible and drug-resistant TB more effectively and safely.

Linezolid (LZD) was the first oxazolidinone approved for treating drug-resistant tuberculosis. A newly approved regimen combining LZD with bedaquiline (BDQ) and pretomanid (PMD) (BPaL regimen) is the first 6-month oral regimen that is effective against multidrug- and extensively drug-resistant tuberculosis. However, LZD toxicity, primarily due to mitochondrial protein synthesis inhibition, may undermine the efficacy of LZD regimens, and oxazolidinones with higher efficacy and lower toxicity during prolonged administration are needed. OTB-658 is an oxazolidinone anti-TB candidate derived from LZD that could replace LZD in TB treatment. We previously found that OTB-658 had better anti-TB activity and safety than LZD in vitro and in vivo . In the present work, two murine TB models were used to evaluate replacing LZD with OTB-658 in LZD-containing regimens. In the C3HeB/FeJ murine model, replacing 100 mg/kg LZD with 50 mg/kg OTB-658 in the BDQ + PMD backbone significantly reduced lung and spleen CFU counts ( P < 0.05), and there were few relapses at 8 weeks of treatment. Replacing 100 mg/kg LZD with 50 or 100 mg/kg OTB-658 in the pyrifazimine (previously called TBI-166) + BDQ backbone did not change the anti-TB efficacy and relapse rate. In BALB/c mice, replacing 100 mg/kg LZD with 100 mg/kg OTB-658 in the TBI-166 + BDQ backbone resulted in no culture-positive lungs at 4 and 8 weeks of treatment, and there were no significant differences in relapses rate between the groups. In conclusion, OTB-658 is a promising clinical candidate that could replace LZD in the BPaL or TBI-166 + BDQ + LZD regimens and should be studied further in clinical trials.

To date, the clinical use of the anti-tubercular therapy bedaquiline has been somewhat limited due to safety concerns. Recent investigations determined that modification of the B- and C-ring units of bedaquiline delivered new diarylquinolines (for example TBAJ-587) with potent anti-tubercular activity yet an improved safety profile due to reduced affinity for the hERG channel. Building on our recent discovery that substitution of the quinoline motif (the A-ring subunit) for C5-aryl pyridine groups within bedaquiline analogues led to retention of anti-tubercular activity, we investigated the concurrent modification of A-, B- and C-ring units within bedaquiline variants. This led to the discovery that 4-trifluoromethoxyphenyl and 4-chlorophenyl pyridyl analogues of TBAJ-587 retained relatively potent anti-tubercular activity and for the 4-chlorophenyl derivative in particular, a significant reduction in hERG inhibition relative to bedaquiline was achieved, demonstrating that modifications of the A-, B- and C-ring units within the bedaquiline structure is a viable strategy for the design of effective, yet safer (and less lipophilic) anti-tubercular compounds., (© 2022 Wiley-VCH GmbH.)

Pretomanid is a nitroimidazole antimicrobial active against drug-resistant Mycobacterium tuberculosis and approved in combination with bedaquiline and linezolid (BPaL) to treat multidrug-resistant (MDR) pulmonary tuberculosis (TB). However, the penetration of these antibiotics into the central nervous system (CNS), and the efficacy of the BPaL regimen for TB meningitis, are not well established. Importantly, there is a lack of efficacious treatments for TB meningitis due to MDR strains, resulting in high mortality. We have developed new methods to synthesize 18 F-pretomanid (chemically identical to the antibiotic) and performed cross-species positron emission tomography (PET) imaging to noninvasively measure pretomanid concentration-time profiles. Dynamic PET in mouse and rabbit models of TB meningitis demonstrates excellent CNS penetration of pretomanid but cerebrospinal fluid (CSF) levels does not correlate with those in the brain parenchyma. The bactericidal activity of the BPaL regimen in the mouse model of TB meningitis is substantially inferior to the standard TB regimen, likely due to restricted penetration of bedaquiline and linezolid into the brain parenchyma. Finally, first-in-human dynamic 18 F-pretomanid PET in six healthy volunteers demonstrates excellent CNS penetration of pretomanid, with significantly higher levels in the brain parenchyma than in CSF. These data have important implications for developing new antibiotic treatments for TB meningitis., (© 2022. The Author(s).)