Abstract Background The increasing incidence of Mycobacterium abscessus ( M. abscessus ) lung infections, together with its intrinsic multidrug resistance, highlights the need for new therapeutic regimens. However, the lack of a reliable chronic infection model in immunocompetent mice limits preclinical evaluation. Methods To mimic the bronchial environment of infected patients, we evaluated the effect of encapsulating M. abscessus in alginate beads on infection progression in BALB/cJRJ mice following intratracheal inoculation, compared with intranasal infection using non-encapsulated bacteria. The impact of dexamethasone treatment (DEX) was also assessed. Bacterial loads in lungs, spleen, liver, and kidneys were quantified over time in untreated and antibiotic-treated mice. Lung inflammation was evaluated by measuring IFN-γ and TNF-α levels. In vitro , the activity of imipenem and bedaquiline was assessed against free or alginate-encapsulated M. abscessus . Results Compared with intranasal infection, intratracheal infection with alginate-encapsulated bacteria resulted in slower pulmonary clearance and greater extrapulmonary dissemination. DEX further enhanced these features, reducing lung clearance, increasing dissemination, and amplifying lung inflammation. Bedaquiline showed no effect, whereas imipenem efficacy depended on treatment timing. For both drugs, alginate encapsulation reduced in vitro antibacterial activity. Conclusion This model represents a step toward a chronic M. abscessus infection model characterized by moderate lungs clearance, extrapulmonary dissemination, and pronounced inflammatory responses. Reduced antibiotic activity against alginate-encapsulated bacteria may more accurately predict treatment efficacy in humans than activity measured against free bacteria.

Tuberculosis (TB) is the worldwide leading infectious killer due to a single pathogen and increasing antimicrobial resistance (AMR) makes it imperative to discover and develop new drugs with novel modes of action (MoAs) to treat TB infections. Phenotypic screening of chemical libraries has proven effective at identifying new compounds against bacterial pathogens. However, a major limitation of standard screens is their inability to uncover the MoA of hits thereby preventing targeted selection of compounds with novel MoAs. Linking drug perturbations to mutants from images could potentially enable to predict the targets of compounds that act through novel MoAs. Here, we develop a deep learning (DL)-based method to screen drug-treated Corynebacterium glutamicum ( Cglu ), a surrogate model for Mycobacterium tuberculosis ( Mtb ). Our DL model is based on a convolutional neural network architecture that takes high throughput images as input and is trained to distinguish between different MoAs. We show that our approach can robustly differentiate between the MoAs of established antibiotics and correctly recognise the MoA of antibiotics that were not previously seen by the DL model. We also show that inhibitors with the same and previously unseen MoA cluster together and apart from all other reference drugs, allowing for new MoA discovery. Importantly, we show that our model links images of chemical (drugs) and genetic (mutants) perturbations targeting similar pathways, thus paving the way towards mutant-based target prediction of compounds that act through novel MoAs, directly from high-content images. Finally, we explore the phenotypes induced by genetic disruption of pathways and demonstrate that features extracted with our DL model recover known biological relationships from high-throughput images alone using the cell cycle of Cglu as a case study, a finding with promising potential for fundamental mechanistic studies.

Abstract The lack of a reliable chronic murine model limits drugs evaluation against Mycobacterium abscessus . Models show discrepancies, especially regarding host factors (mouse strain, sex and age). Using beads-model, we compared BALB/cJRJ and C57BL/6NCrl across sexes and ages. BALB/cJRJ showed more sustained infection and lower variability, with no significant sex- or age-related differences. Considering these results and the higher prevalence of NTM pulmonary infections in female patients, 5-6 weeks-old female BALB/cJRJ are appropriate for M. abscessus beads-model.

DNA gyrase is an essential bacterial enzyme and a clinically validated target for the treatment of tuberculosis. However, the discovery of new inhibitors remains limited by the many challenges regarding the manipulation on pathogenic mycobacteria. This study validates Corynebacterium glutamicum (Cglu) as a safe, non-pathogenic surrogate for Mycobacterium tuberculosis (Mtb) to investigate DNA gyrase and facilitate the identification of new inhibitors. Using Cglu as a target allows for fast whole-cell screening under safe conditions while ensuring efficient drug uptake. Cglu shares key physiological features with Mtb, including genome size, complex cell wall structure, and a single type I and type II topoisomerase. Structural and functional comparisons emphasize the similarity of Cglu and Mtb gyrases, which share 70% sequence identity and show comparable catalytic properties and responsiveness to known inhibitors. Thus, the cryo-EM structure of the Cglu gyrase-DNA complex at 3.2 Å resolution reveals highly conserved drug-binding pockets for known anti-gyrase inhibitors and the genetic depletion of gyrA or gyrB in Cglu causes severe growth and morphological defects, mirroring the effects of chemical inhibition and allowing to link gyrase function to cellular phenotypes. Comparative imaging of different inhibitor classes (fluoroquinolones, aminocoumarins, NBTIs) uncovers distinct morphological signatures that reflect each compound's mode of action. Finally, cross-species complementation confirms functional conservation but also highlights subtle structural differences affecting efficiency. Together, these findings establish Cglu as a robust and biosafe model for dissecting gyrase function, visualizing DNA topology dynamics, and accelerating the discovery of gyrase-targeting antimicrobials. More generally, our studies demonstrate the feasibility of using Cglu as a cell-based screening platform to discover new anti-tuberculous compounds targeting conserved mechanisms, not only for validated TB drug targets such as DNA gyrase but also for new, yet to be identified, targets.

Carbapenemase-producing Enterobacterales (CPE) present limited therapeutic options. Optimal treatment requires identifying the carbapenemase type, often requiring confirmatory testing beyond routine susceptibility results. We develop MALCA, a machine-learning classifier that uses routine disc diffusion antibiogram results to directly detect CPE and identify the carbapenemase type. From 11,992 clinical isolates, we build a stepwise random-forest pipeline and derive two classifiers based on panels of 22 or 8 antibiotics (MALCA-22 and MALCA-8). In an external validation study involving 8514 isolates, both MALCA classifiers achieved sensitivity and specificity >96% for CPE detection, outperforming European and French algorithms developed for CPE screening. For the most prevalent carbapenemases, MALCA achieve sensitivities exceeding 97% and specificities above 98%, particularly for OXA-48-like, NDM, and KPC producers. MALCA is a rapid, and inexpensive diagnostic tool that uses solid antibiogram data to detect and type CPE, enabling earlier targeted therapy and diagnostic guidance without additional reagents or human resources.

ABSTRACT Leprosy treatment requires prolonged therapy with challenging patient follow-up. New regimens are needed to simplify current treatments. A recent clinical trial evaluating bedaquiline has shown promising results; however, to prevent the emergence of drug resistance, additional therapeutic options are required. Telacebec (TCB), an imidazopyridine amide targeting the Mycobacterium leprae electron transport chain, represents a promising candidate. In this study, we determined the minimal effective dose (MED) of TCB against M. leprae in vivo by using the proportional bactericidal method in the mouse footpad model. Results were analyzed by using microscopy, RLEP qPCR and molecular viability. The MED obtained was 20 mg/kg. TCB doses ≥20 mg/kg achieved complete bacterial clearance, similar to bedaquiline 25mg/kg. Molecular enumeration confirmed these findings whereas molecular viability assessment had limited applicability due to insufficient bacterial burden. These findings provide a strong foundation for clinical trial design and the development of combination therapies.

Ethionamide (Eto) and prothionamide (Pto) are second-line antibiotics used for tuberculosis (TB) treatment. Both are prodrugs whose antibacterial activity depends on bioactivation by oxidases in Mycobacterium tuberculosis, including the Baeyer-Villiger monooxygenase MymA. Through biophysical, genetic, and cellular assays, we show that the clinical candidate alpibectir (Alp, BVL-GSK098) binds the transcriptional regulator VirS, increasing MymA expression and potentiating Eto and Pto activity. Alpibectir also boosts the activity of the corresponding host-derived sulfoxide metabolites. We additionally show that alpibectir exhibits intrinsic antibacterial activity via overexpression of the mymA operon. The alpibectir/Eto (AlpE) combination is rapidly bactericidal in vitro and in mice, lowers the frequency of spontaneous resistance of Eto, and remains active on Eto- and isoniazid-resistant strains, including isolates with inhA promoter mutations. Alpibectir was safe in a Phase 1 human clinical trial. Together with the potentiation data presented here, these findings highlight its potential to optimize TB chemotherapy by reducing Eto/Pto doses, which can minimize dose-related side effects, enhancing adherence.

Multidrug-resistant (MDR) strains of Mycobacterium tuberculosis represent an obstacle to eradicating tuberculosis (TB) due to the low treatment success rate of MDR TB. Among them, the MDR B0/W148 clone has recently evolved from the M. tuberculosis Beijing lineage 2 and is widely disseminated in Russia and Europe. To get more insights into the genetic factors underlying the evolutionary success of the MDR M. tuberculosis B0/W148 clone in addition to environmental and patient-related features, we focused on two mutations specific to this clone that are found in the transcriptional regulators WhiB6 and KdpDE and investigated in a H37Rv strain background the transcriptional profile associated with these mutations and their impact on the in vitro and in vivo growth characteristics. Through the construction and use of H37Rv∆whiB6, H37Rv∆kdpDE, and complemented strains, neither mutation impaired the in vitro growth of M. tuberculosis in standard mycobacterial growth media. The mutation T51P in whiB6 prevented the upregulation of 9 genes in the esx-1 core region and 44 genes elsewhere in the genome, while the deletion of two nucleotides in kdpD leads to a fusion protein of KdpD with KdpE that inhibits the transcriptional activity of KdpE. Neither mutation led to hypervirulence in a mouse infection model. These results point to the role of other MDR B0/W148 specific mutations in the wide geographic diffusion of this clone and/or put in question a hypothesized hypervirulence as a driving factor for this large dissemination. IMPORTANCE:Human tuberculosis (TB), caused by the bacterium Mycobacterium tuberculosis, remains a global public health issue estimated to have been responsible for 1.25 million deaths in 2023. Multidrug-resistant (MDR) strains of M. tuberculosis, resistant to rifampicin and isoniazid, lead to lower treatment success. Among them, the MDR B0/W148 clone has widely disseminated in Russia and Europe. To get more insights into the genetic factors underlying the evolutionary success of this clone, we investigated two strain-specific mutations found in the transcriptional regulators WhiB6 and KdpDE. By constructing and analyzing laboratory M. tuberculosis strains carrying these specific mutations, we found numerous changes in their transcriptional profiles, whereas we observed only a little impact of these mutations on the virulence of M. tuberculosis in a mouse infection model. Our study provides new insights into the transcriptional landscape of the selected MDR strains, although no direct connection to virulence could be established.

The reported poor treatment outcomes for extensively drug-resistant tuberculosis (TB) in the European region highlight the urgent need for effective and context-appropriate diagnostic strategies. While the World Health Organisation (WHO) provides model algorithms, these require adaptation to the European Union/European Economic Area (EU/EEA) context, a setting with low TB incidence but high resources. This viewpoint from the European Reference Laboratory Network for TB (ERLTB-Net) proposes a tailored diagnostic algorithm that prioritises the universal use of WHO-recommended molecular rapid diagnostic tests, systematic culture, and whole genome sequencing (WGS). This approach integrates phenotypic drug susceptibility testing strategically and outlines the possible role of targeted next-generation sequencing (tNGS) in the EU/EEA setting. The algorithm also addresses the importance of diagnostic harmonisation, cross-border collaboration, and sustained investment in sequencing capacity. By aligning diagnostic practices with the regional epidemiology and laboratory infrastructure, this stepwise, resource-sensitive approach aims to strengthen TB control, improve treatment outcomes, and guide public health action in the EU/EEA.

Tuberculosis (TB) remains the foremost cause of death from infectious diseases globally, prompting ongoing efforts to improve treatment options. This includes developing compounds with novel modes of action and identifying optimal treatment regimens that allow for treatment shortening. One promising strategy involves targeting cytochrome bc1 oxidase in Mycobacterium tuberculosis, a key enzyme in the respiratory chain. In this study, we evaluate the potential of cytochrome bc1 inhibitors as partner drugs in TB combination regimens. Using a relapsing mouse model, we demonstrate that these inhibitors enhance regimen sterilisation and significantly reduce the time required for effective treatment. We also propose several novel combination strategies for both multidrug-resistant and drug-sensitive TB, where cytochrome bc1 inhibitors contribute to sterilisation and improved treatment outcomes. Furthermore, M. tuberculosis clinical isolates exhibited heightened susceptibility to cytochrome bc1 inhibitors compared to laboratory-adapted strains, highlighting the importance of using clinical isolates in TB drug discovery to better reflect the diversity of TB populations. These findings emphasise the potential of cytochrome bc1 inhibition in the development of more effective and shorter treatment regimens for TB, supporting the need for further clinical investigation.

The rise of multidrug-resistant tuberculosis (TB) has increased the need for new antitubercular (anti-TB) drugs and the identification of novel drug targets. One promising target is Mycobacterium tuberculosis (Mtb) cytochrome P450 enzymes (P450s). This study focuses on the characterization of CYP135B1, a prevalent Mtb P450. Using a combination of microbiology, genomics, bioinformatics, docking, spectroscopy, and mass spectrometry, researchers successfully expressed, purified, and characterized CYP135B1. A 3D model was built with AlphaFold 3. The enzyme displayed typical features of P450 proteins and showed strong binding to imidazole derivatives. Notably, CYP135B1 metabolized the anti-TB drug SQ109 by inserting oxygen into its geranyl moiety in a manner distinct from CYP124A1. However, genetic studies using a ΔCYP135B1 mutant strain revealed that CYP135B1 is not required for SQ109's antibacterial activity, as its deletion did not affect drug efficacy despite CYP135B1 metabolizes SQ109.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has transformed microbiology by enabling rapid and cost-effective pathogen identification. However, differentiating between closely related species remains challenging. Current analytical approaches, such as database-driven approaches, still underutilize the information contained in mass spectra. In response to this challenge, we developed MSclassifR, an R package designed to facilitate the construction of data analysis pipelines for accurate mass spectra classification using machine learning (ML) techniques. MSclassifR provides end-to-end pipelines tailored for microbiological diagnostics, covering preprocessing, mass-to-charge (m/z) selection, and classification of mass spectra. One of the main strengths of the package is its m/z selection method based on random forest (RF) variable importance, which improves identification accuracy by focusing on the most informative spectral features. We assessed classification pipelines constructed using MSclassifR through rigorous experiments conducted on diverse datasets that included bacterial species or subspecies and virulent/avirulent phenotypes, illustrating the package's versatility across diverse applications. Moreover, the pipelines achieved high accuracy when applied to SARS-CoV-2 nasal swab mass spectra acquired using various MALDI-TOF instruments. Comparisons of multiple pipelines revealed that RF-based pipelines achieved the best performances on various-sized datasets. The MSclassifR package offers microbiologists an open-source solution that leverages ML to enhance MALDI-TOF MS's diagnostic capabilities. It is available for download from the Comprehensive R Archive Network (http s://cran.r-project.org/web/packages/MSclassifR/).

High-dose isoniazid is recommended to treat multidrug-resistant tuberculosis (MDR TB). Among 958 MDR TB isolates identified in France during 2008-2022, 93.1% exhibited high-level isoniazid resistance, and molecular testing showed limited diagnostic accuracy in predicting resistance. Clinicians should reconsider using high-dose isoniazid in MDR TB treatment because of suboptimal effect and toxicity concerns.

Drug-resistant tuberculosis (TB) continues to challenge treatment options, necessitating the exploration of new compounds of novel targets. The mycobacterial respiratory complex cytochrome bc1:aa3 has emerged as a promising target, exemplified by the success of first-in-class inhibitor Q203 in phase 2 clinical trials. However, to fully exploit the potential of this target and to identify the best-in-class inhibitor more compounds need evaluation. Here, we introduce JNJ-2901, a novel Q203 analogue, that demonstrates activity against multidrug-resistant M. tuberculosis clinical strains at sub-nanomolar concentration and 4-log reduction in bacterial burden in a mouse model of TB infection. Inhibitory studies on purified enzymes validate the nanomolar inhibitions observed in mycobacterial cells. Additionally, cryo-EM structure analysis of cytochrome bc1:aa3 bound to JNJ-2901 reveals the binding pocket at the menaquinol oxidation site (Qp), akin to other substate analogue inhibitors like Q203 and TB47. Validation of the binding site is further achieved by generating and isolating the JNJ-2901 resistant mutations in M. tuberculosis, followed by purification and resistance analysis of the resistant cytochrome bc1:aa3 complex. Our comprehensive work lays the foundation for further clinical validations of JNJ-2901.

BackgroundNew drugs targeting the electron transport chain (ETC) seem to be a promising advance in leprosy treatment. In this study, we evaluated the bactericidal activity of telacebec (TCB), a phase 2 drug candidate for tuberculosis, alongside known ETC-targeting antibiotics, bedaquiline (BDQ) and clofazimine (CFZ), as monotherapy or in combination.Methodology/ principal findingsWe used the reference leprosy proportional bactericidal mouse footpad model. Four hundred and ten mice were inoculated in the footpads with 5x104 to 5x100 bacilli of M. leprae strain THAI53 for the untreated control group and groups treated with drug-monotherapies, and with 5x104 to 5x101 for groups treated with drug-combinations. Mice were randomly allocated into the following groups: 2 control groups (untreated or standard multi drug therapy (MDT), rifampin, dapsone and clofazimine with dosing equipotent to human dosing) and 7 test groups (TCB 10mg/kg, bedaquiline 25mg/kg (BDQ), clofazimine 20mg/kg (CFZ), CFZ + BDQ, TCB + BDQ, TCB + CFZ, TCB + CFZ + BDQ). Mice in the test groups received either one month treatment (MDT) or a single dose of the drugs (TCB, RIF, BDQ, CFZ). Twelve months later, mice were sacrificed to enumerate M. leprae bacilli in the footpad. All the footpads became negative in the MDT, TCB and combination groups except in the TCB + CFZ group where 2 mice remained positive in the 5x104 inoculum.ConclusionWe demonstrated that monotherapy of TCB exhibited bactericidal activity against M. leprae comparable to that of MDT and that all combination therapies were as effective as MDT, except the combination TCB + CFZ, possibly due to an antagonism between these two drugs.

OBJECTIVES:Nontuberculous mycobacteria (NTM) bone and joint infections (BJIs) are uncommon. We evaluated the characteristics of BJIs and identified differences according to immune status. METHODS:We performed a multicenter retrospective study in France involving patients with documented NTM BJI over a 9-year period. We collected the clinical and microbiological characteristics, management, and clinical outcomes of the patients. RESULTS:Overall, 95 patients were included, of whom 50.5% (48/95) were immunosuppressed. Tenosynovitis was more frequent in the immunocompetent group, and native arthritis more common in the immunosuppressed group. Mycobacerium marinum and M. abscessus complex were significantly more frequent in the immunocompetent group, and M. avium and M. xenopi were significantly more frequent in the immunosuppressed group. The combination of antibiotherapy with surgery tended to be more frequent in the immunocompetent than the immunosuppressed group (63.8% (30/47) vs 47.8% (22/46), respectively); of the latter, 45.7% (21/46) received antimicrobial therapy alone, a higher frequency than in the immunocompetent group (23.4%, 11/47). The median duration of antimicrobial treatment was similar in the two groups (11 months). Mortality was significantly higher in the immunosuppressed group. CONCLUSIONS:Although the clinical presentations and the NTM species involved in BJI differed according to immune status, most recovered completely after treatment.

Clostridioides difficile (CD) infections are defined by toxins A (TcdA) and B (TcdB) along with the binary toxin (CDT). The emergence of the 'hypervirulent' (Hv) strain PR 027, along with PR 176 and 181, two decades ago, reshaped CD infection epidemiology in Europe. This study assessed MALDI-TOF mass spectrometry (MALDI-TOF MS) combined with machine learning (ML) and Deep Learning (DL) to identify toxigenic strains (producing TcdA, TcdB with or without CDT) and Hv strains. In total, 201 CD strains were analysed, comprising 151 toxigenic (24 ToxA(+)B(+)CDT(+), 22 ToxA(+)B(+)CDT(+) Hv(+) and 105 ToxA(+)B(+)CDT(-)) and 50 non-toxigenic (ToxA(-)B(-)) strains. The DL-based classifier exhibited a 0.95 negative predictive value for excluding ToxA(-)B(-) strains, showcasing accuracy in identifying this strain category. Sensitivity in correctly identifying ToxA(+)B(+)CDT(-) strains ranged from 0.68 to 0.91. Additionally, all classifiers consistently demonstrated high specificity (>0.96) in detecting ToxA(+)B(+)CDT(+) strains. The classifiers' performances for Hv strain detection were linked to high specificity (>= 0.96). This study highlights MALDI-TOF MS enhanced by ML techniques as a rapid and cost-effective tool for identifying CD strain virulence factors. Our results brought a proof-of-concept concerning the ability of MALDI-TOF MS coupled with ML techniques to detect virulence factor and potentially improve the outbreak's management.

Recent efforts to improve tuberculosis (TB) treatment options have focused on developing molecules with novel mechanisms of action and identifying optimal treatment regimens. Inhibition of Mycobacterium tuberculosis cytochrome bc1 oxidase has emerged as a promising therapeutic target that could potentially contribute to improved TB combination regimens. Using a relapsing mouse model, we demonstrate that cytochrome bc1 inhibitors could serve as effective partner drugs, enhancing regimen sterilisation. We propose several novel regimen strategies for both multidrug-resistant TB (MDR-TB) and drug-sensitive TB (DS-TB), where cytochrome bc1 inhibitors contribute to sterilisation and treatment shortening. Additionally, we show that clinical isolates exhibit heightened susceptibility to cytochrome bc1 inhibitors compared to laboratory-adapted strains, further supporting their translational potential. These findings suggest that cytochrome bc1 inhibitors have significant potential to improve TB treatment outcomes and highlight the need for further studies to evaluate their clinical contribution to novel treatment regimens.

ADVANCES IN ANTIBIOTIC THERAPY FOR TUBERCULOSIS. Treatment of tuberculosis is experiencing significant advancements. For the first time, a therapeutic regimen based on rifapentine and moxifloxacin allows for a reduction of treatment duration of drug-susceptible tuberculosis from 6 to 4 months. Regarding multidrug-resistant tuberculosis, combinations of new antituberculosis drugs (bedaquiline, linezolid, delamanid/pretomanid, moxifloxacin) have the potential to reduce the treatment duration from 20 to 6 months. Additionally, considering the extent of anatomical involvement and bacterial burden allows for strategies that involve variable treatment durations based on the severity of the disease. The new tuberculosis treatments thus appear to be shorter and more personalized.