Nicolas Veziris

To sustain the anti-tuberculosis drug development pipeline, novel antibiotics must be developed both as standalone agents and as components of future combination regimens. To this end, tricyclic-spirolactams (TriSLa)-based inhibitors of mycobacterial type II NADH dehydrogenase (Ndh-2) represent a promising novel class of antibiotics, though activity is carbon source dependent. Initial studies established an enhanced understanding of the impact of TriSLa on mycobacteria using metabolomics and transcriptomics. Then, CRISPRi chemical-genetics and extensive in vitro TriSLa-combination studies showed that Ndh-2 inhibition resulted in rendering other components of the electron transport chain vulnerable, even in conditions when TriSLa was inactive alone. Finally, identified in vitro synergistic TriSLa-antibiotic combinations were validated to also give modestly improved in vivo efficacy. Together, this work expands our understanding of how Ndh-2 inhibition impacts bacterial physiology and reveals how it sensitizes the bacterium to electron transport chain inhibitors, providing a foundation for future TriSLa-based combination regimens.

The lack of a reliable chronic murine model limits drug evaluation against Mycobacterium abscessus . Existing models show discrepancies, especially regarding host factors such as mouse strain, sex, and age. To address this issue, we compared infection establishment and progression in BALB/cJRJ and C57BL/6NCrl mice across sexes and ages using a clinically relevant alginate bead-based model that mimics biofilm-like infection. BALB/cJRJ mice showed more sustained infection and lower variability, with no significant sex- or age-related differences. Based on these results, and taking into account the higher prevalence of nontuberculous mycobacterial pulmonary infections in female patients, as well as practical considerations, 5- to 6-week-old female BALB/cJRJ mice are appropriate for the M. abscessus bead model. IMPORTANCE: Nontuberculous mycobacteria (NTM), including Mycobacterium abscessus , are increasingly recognized as opportunistic pathogens in humans. The urgent need for new anti- M. abscessus therapies is hindered by the lack of standardized and reproducible chronic murine infection models, as well as by discrepancies in the literature regarding the influence of host factors (mouse strain, sex, and age). Here, we systematically compared lung infections in the two most commonly used immunocompetent mouse strains, BALB/cJRJ and C57BL/6NCrl, across sexes and two age groups (5–6 vs 8–10 weeks), using the alginate-beads infection model. We identified 5-to 6-week-old female BALB/c mice as the most suitable conditions for implementing the M. abscessus beads model, providing more sustained infection and lower variability. Our findings clarify the impact of host-related variables and provide guidance for improving the standardization and reproducibility of murine models used in preclinical evaluation of anti-NTM therapies.

Macrolides are key agents for treating infections caused by non-tuberculous mycobacteria (NTM). Nevertheless, chromosomal erm genes conferring inducible macrolide resistance are described in some NTM species, such as Mycobacterium abscessus and M. fortuitum , whereas M. chelonae had long been considered as lacking a functional e rm . Recent descriptions from the USA and Japan of a new plasmid-borne erm (55) ( erm (55) ^P ) in M. chelonae and other rapidly growing mycobacteria (RGM) have challenged this assumption. We investigated erm (55) ^P occurrence in clinical RGM referred to the French National Reference Centre for Mycobacteria between 2012 and 2026 by genome screening and erm (55) ^P specific real-time PCR. Positive isolates underwent long-read whole genome sequencing (GridIon, Oxford Nanopore Technologies). Clarithromycin (CLR) minimum inhibitory concentration (MIC) was determined by broth microdilution (RAPMYCO and FRATMYC, Thermo Fisher) and read up to 14 days. Five clinical isolates showing inducible CLR resistance (MIC range 128 mg/L on day 14) were positive for erm (55) ^P : one M. chelonae , three M. neoaurum , and one M. parafortuitum . erm (55) ^P -positive M. chelonae genomes from this and previous descriptions did not cluster together in the phylogenetic analysis of 263 genomes. The assembled plasmids showed high similarity to previously reported erm (55)-carrying plasmids, especially within the erm (55) ^P region. The upstream sequence of erm (55) ^P showed a secondary structure compatible with a possible translation attenuation mechanism. These findings document the first report of a plasmid-borne erm (55) in Europe in M. chelonae and other RGM and raise concern about the emergence of plasmid macrolide resistance in NTM.

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.

Occupational Transmission of Extensively Drug-Resistant Tuberculosis, France

Background: Identifying patients at risk of nontuberculous mycobacterial pulmonary disease (NTM-PD) is challenging. Delays in NTM-PD identification and management are associated with declining lung function and increased morbidity and mortality.Study design and methods: European NTM-PD experts (n=12) participated in a three-round modified Delphi process to score symptoms and comorbidities potentially associated with NTM-PD as reasons to test for nontuberculous mycobacteria.Results: Experts reached a consensus on the symptoms and comorbidities that should and should not prompt testing for nontuberculous mycobacteria. Requirements for testing were scored as high (mean ≥7), medium (mean ≥4–<7) or low (mean <4). Nontuberculous mycobacteria testing should be undertaken when multiple suggestive symptoms are present simultaneously in all patients except those with cancer (7.3–8.8), or when radiology is indicative of NTM-PD (≥8.9). Symptoms of persistent sputum production, recurrent respiratory infection and haemoptysis should prompt testing for nontuberculous mycobacteria, particularly in those with underlying respiratory diseases. Symptomatic patients with bronchiectasis or previous tuberculosis/NTM-PD or those being prescribed or undergoing long-term macrolide therapy for a respiratory condition should also be tested. Testing is not warranted in patients without an underlying respiratory disorder or in those without a history of respiratory disorders unless presenting with multiple symptoms.Conclusions: Assessing patients’ risk of NTM-PD is challenging. This Delphi consensus process provides insight into symptoms and clinical characteristics that should prompt NTM-PD assessment. Timely testing and diagnosis would enable initiation of appropriate management.

Tweetable abstract How to identify MAC-PD patients with limited treatment options: an expert consensushttps://bit.ly/3QwLQ8T

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.