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Background: Non-tuberculous mycobacteria (NTM) are a diverse group of environmental bacteria that are increasingly associated with human infections and difficult to treat. Plasmids, which might carry resistance and virulence factors, remain largely unexplored in NTM., Methods: We used publicly available complete genome sequence data of 328 NTM isolates belonging to 125 species to study gene content, genomic diversity, and clusters of 196 annotated NTM plasmids. Furthermore, we analyzed 3755 draft genome assemblies from over 200 NTM species and 5415 short-read sequence datasets from six clinically relevant NTM species or complexes including M. abscessus, M. avium complex, M. ulcerans complex and M. kansasii complex, for the presence of these plasmids., Results: Between one and five plasmids were present in approximately one-third of the complete NTM genomes. The annotated plasmids varied widely in length (most between 10 and 400 kbp) and gene content, with many genes having an unknown function. Predicted gene functions primarily involved plasmid replication, segregation, maintenance, and mobility. Only a few plasmids contained predicted genes that are known to confer resistance to antibiotics commonly used to treat NTM infections. Out of 196 annotated plasmid sequences, 116 could be grouped into 31 clusters of closely related sequences, and about one-third were found across multiple NTM species. Among clinically relevant NTM, the presence of NTM plasmids showed significant variation between species, within (sub)species, and even among strains within (sub)lineages, such as dominant circulating clones of Mycobacterium abscessus., Conclusions: Our analysis demonstrates that plasmids are a diverse and heterogeneously distributed feature in NTM bacteria. The frequent occurrence of closely related putative plasmid sequences across different NTM species suggests they may play a significant role in NTM evolution through horizontal gene transfer at least in some groups of NTM. However, further in vitro investigations and access to more complete genomes are necessary to validate our findings, elucidate gene functions, identify novel plasmids, and comprehensively assess the role of plasmids in NTM., Competing Interests: Declarations. Ethics approval and consent to participate: Not applicable. Consent for publication: Not applicable. Competing interests: MD, NW, IB, VD, CU, MM, and SN have no conflicts of interest. FPM is an employee of Basilea Pharmaceutica, Allschwil, Switzerland. FPM contributed to the work presented above outside of this employment. Basilea Pharmaceutica played no role in the conceptualization, conduction, or financing of this study., (© 2025. The Author(s).)

Objectives: Extrapulmonary tuberculosis (EPTB) presents with nonspecific symptoms that can pose a significant diagnostic challenge. Various factors, including age, sex, and HIV status, have been associated with an increased risk of developing EPTB. However, the influence of the lineage of the infecting Mycobacterium tuberculosis complex (Mtbc) strain remains controversial., Methods: Between 2008 and 2023, comprehensive clinical data from 1035 cases, along with whole genome sequencing data of the respective Mtbc strains, have been collected. To examine the association between Mtbc lineage and EPTB, we calculated crude and adjusted OR using logistic regression and performed propensity score matching with a subset of the cohort., Results: Of the 1035 patients, 272 had exclusively extrapulmonary disease and 138 had both pulmonary and extrapulmonary disease. Patients infected with a lineage 1 strain had the highest odds of developing EPTB in the univariate analysis (OR, 3.30; 95% CI, 1.97-5.49). However, Mtbc lineage was not a significant predictor in the multivariable model, whereas the odds of developing extrapulmonary disease were higher among patients born in the South-East Asian Region (adjusted OR, 6.00; 95% CI, 3.41-10.69) and the Eastern Mediterranean Region (adjusted OR, 5.95; 95% CI, 3.61-9.96) compared with those born in the European Region. Furthermore, female sex and age were significant positive predictors for EPTB., Discussion: Our results demonstrate that host factors, such as geographic origin, age, and sex are stronger predictors for EPTB than infection with an Mtbc strain of a particular lineage. Further investigation of this host-pathogen interaction is needed., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Objectives: While the reported incidence of non-tuberculous mycobacterial (NTM) infections is increasing, the true prevalence remains uncertain due to limitations in diagnostics and surveillance. The emergence of rare and novel species underscores the need for characterization to improve surveillance, detection, and management., Methods: We performed whole-genome sequencing (WGS) and/or targeted deep-sequencing using the Deeplex Myc-TB assay on all NTM isolates collected in Slovakia and the Czech Republic between the years 2019 to 2023 that were unidentifiable at the species level by the routine diagnostic line probe assays (LPA) GenoType CM/AS and NTM-DR. Minimal inhibitory concentrations against amikacin, ciprofloxacin, moxifloxacin, clarithromycin, and linezolid were determined, and clinical data were collected., Results: Twenty-eight cultures from different patients were included, of which 9 (32.1%) met the clinically relevant NTM disease criteria. The majority of those had pulmonary involvement, while two children presented with lymphadenitis. Antimycobacterial resistance rates were low. In total, 15 different NTM species were identified, predominantly rare NTM like M. neoaurum, M. kumamotonense and M. arupense. Notably, clinically relevant M. chimaera variants were also identified with WGS and Deeplex-Myc TB, which, unlike other M. chimaera strains, appeared to be undetectable by LPA assays. Deeplex detected four mixed infections that were missed by WGS analysis. In contrast, WGS identified two novel species, M. celatum and M. branderi, which were not detected by Deeplex-Myc TB. Importantly, one of these novel species strains was associated with clinically relevant pulmonary disease., Discussion: Our study demonstrates the clinical relevance of uncommon NTM and the effectiveness of targeted deep-sequencing combined with WGS in identifying rare and novel NTM species., Competing Interests: Declarations. Ethical approval: This study was approved by the Ethical Committee of the Jessenius Faculty of Medicine in Martin, Comenius University Bratislava (EK65/2021). All methods were carried out following relevant guidelines and regulations. Consent for publication: Not applicable. Content of the publication: During the preparation of this work the author(s) used chatgpt in order to enhance the clarity and readability of the text. After using this tool/service, the author(s) reviewed and edited the content as needed and take(s) full responsibility for the content of the publication. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Background: There are few data on the treatment of children and adolescents with multidrug-resistant (MDR) or rifampicin-resistant (RR) tuberculosis, especially with more recently available drugs and regimens. We aimed to describe the clinical and treatment characteristics and their associations with treatment outcomes in this susceptible population., Methods: We conducted a systematic review and individual participant data meta-analysis. Databases were searched from Oct 1, 2014, to March 30, 2020. To be eligible, studies must have included more than five children or adolescents (0-19 years of age) treated for microbiologically confirmed or clinically diagnosed MDR or RR tuberculosis within a defined treatment cohort, and reported on regimen composition and treatment outcomes. Abstracts were screened independently by two authors to identify potentially eligible records. Full texts were reviewed by two authors independently to identify studies meeting the eligiblity criteria. For studies meeting eligiblity criteria, anonymised individual patient data was requested and individiual level data included for analysis. The main outcome assessed was treatment outcome defined as treatment success (cure or treatment completed) versus unfavourable outcome (treatment failure or death). Multivariable logistic regression models were used to identify associations between clinical and treatment factors and treatment outcomes. This study is registered with Prospero (CRD42020187230)., Findings: 1417 studies were identified through database searching. After removing duplicates and screening for eligibility, the search identified 23 369 individual participants from 42 studies, mostly from India and South Africa. Overall, 16 825 (72·0%) were successfully treated (treatment completed or cured), 2848 died (12·2%), 722 (3·1%) had treatment failure, and 2974 (12·7%) were lost to follow-up. In primary analyses, the median age was 16 (IQR 13-18) years. Of the 17 764 (87·1%) participants with reported HIV status, 2448 (13·8%) were living with HIV. 17 707 (89·6%) had microbiologically confirmed tuberculosis. After adjusting for significant factors associated with treatment outcome, the use of two (adjusted odds ratio [OR] 1·41 [95% CI 1·09-1·82]; p=0·008) or three (2·12 [1·61-2·79]; p<0·0001) WHO-classified group A drugs (bedaquiline, moxifloxacin, levofloxacin, and linezolid) compared with the use of no group A drugs at all was positively associated with treatment success., Interpretation: Younger and clinically diagnosed children are underrepresented among those treated for MDR and RR tuberculosis and should be a focus for case-finding efforts. Overall treatment outcomes in our analysis were better than in adults but lower than the international targets of 90% or more individuals successfully treated. Treatment with more group A drugs was associated with better treatment outcomes in children and adolescents, highlighting the need for more rapid access to these drugs and improved regimens., Funding: Unitaid., Competing Interests: Declaration of interests AJG-P reports that his institution receives grant funding from the US National Institutes of Health (NIH) and Unitaid for paediatric studies of bedaquiline, delamanid, pretomanid, clofazimine, moxifloxacin, and levofloxacin. J-JY reports donations of linezolid (Zyvox) from Pfizer, Delamanid (Deltyba) from Otsuka Pharmaceutical, and Rifampicin (Rifampcin) from Yuhan for the for the clinical trials, in which he served as a principal investigator. JF reports grant funding form the Stop TB Partnership's Global Drug Facility. JRC reports grant funding to his institution from the Canadian Institutes of Health, Fonds de recherche du Quebec-Sante, the National Sanatorium Association, and WHO; and consulting fees from WHO, the World Bank, and the Saskatchewan Auditors Office. BSG reports grant funding from the Carlos III Institute of Health, Ministry of Economy and Competitiveness (Spain). GEV reports grant funding from the NIH, US Agency for International Development (USAID), Unitaid, Medecins Sans Frontiers, and Partners in Health; and consulting fees from the Gates Medical Research Institute. RS reports grant funding from the NIH, the Bill and Melinda Gates Foundation, USAID, and from UNITE4TB (Academia and Industry United Innovation and Treatment for Tuberculosis). AS reports participating at the Research Lead at UK Academics and Professionals to end Tuberculosis. All other authors declare no competing interests., (Copyright © 2025 World Health Organization. Published by Elsevier Ltd. 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This paper presents an update on the content, accessibility and analytical tools of the EnteroBase platform for web-based pathogen genome analysis. EnteroBase provides manually curated databases of genome sequence data and associated metadata from currently >1.1 million bacterial isolates, more recently including Streptococcus spp. and Mycobacterium tuberculosis, in addition to Salmonella,Escherichia/Shigella,Clostridioides,Vibrio,Helicobacter,YersiniaandMoraxella. We have implemented the genome-based detection of antimicrobial resistance determinants and the new bubble plot graphical tool for visualizing bacterial genomic population structures, based on pre-computed hierarchical clusters. Access to data and analysis tools is provided through an enhanced graphical user interface and a new application programming interface (RESTful API). EnteroBase is now being developed and operated by an international consortium, to accelerate the development of the platform and ensure the longevity of the resources built. EnteroBase can be accessed at https://enterobase.warwick.ac.uk as well as https://enterobase.dsmz.de., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)