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Available drugs are largely ineffective due to an exquisite intrinsic resistance, making Mab infections only comparable to multidrug-resistant tuberculosis. Current treatment is based on lengthy multidrug therapy, complicated by poor outcomes and high rates of treatment failure, recurrence, and mortality. Thus, finding new and more efficient drugs to combat this pathogen is urgent. However, drug discovery efforts targeting Mab have been limited, and traditional drug screening methods are labor-intensive, low-throughput, and do not reflect clinical effectiveness. Therefore, this work aimed to develop a new, efficient, and reliable tool for drug screening against Mab that can be used\n              <jats:italic>in vitro<\/jats:italic>\n              for identifying hits in a high-throughput manner and\n              <jats:italic>in vivo<\/jats:italic>\n              to select drug candidates for future clinical trials. We engineered two stable double-reporter strains of Mab capable of emitting strong fluorescent and luminescent signals. This is due to the expression of mScarlet protein and luciferase enzyme or the entire lux operon. Importantly, these strains maintain the same ground characteristics as the non-transformed Mab strain. We show that these new strains can be applied to various setups, from MIC determination in broth cultures and macrophage infection assays to\n              <jats:italic>in vivo<\/jats:italic>\n              infection (using the\n              <jats:italic>Galleria mellonella<\/jats:italic>\n              model). Using these strains enhances the potential for high-throughput screening of thousands of compounds in a fast and reliable way.\n            <\/jats:p>\n          <\/jats:sec>\n          <jats:sec>\n            <jats:title>IMPORTANCE<\/jats:title>\n            <jats:p>\n              <jats:italic>Mycobacterium abscessus<\/jats:italic>\n              (Mab) is currently considered an \u201cincurable nightmare.\u201d Its intrinsic resistance, high toxicity, long duration, and low cure rates of available therapies often lead to the clinical decision not to treat. Moreover, one of the significant drawbacks of anti-Mab drug development is the lack of correlation between\n              <jats:italic>in vitro<\/jats:italic>\n              susceptibility and clinical efficacy. Most drug screening assays are performed on Mab growing in liquid cultures. But being an intracellular pathogen, inducing granulomas and biofilm formation, the broth culture is far from ideal as\n              <jats:italic>in vitro<\/jats:italic>\n              drug-testing setup. This study presents new double-reporter Mab strains that allow direct real-time bacterial detection and quantification in a non-invasive way. These strains can be applied to an extensive range of experimental settings, far surpassing the utility of single-reporter bacteria. They can be used in all steps of the pre-clinical anti-Mab drug development pipeline, constituting a highly valuable tool to increase its success.\n            <\/jats:p>\n          <\/jats:sec>","DOI":"10.1128\/spectrum.00362-24","type":"journal-article","created":{"date-parts":[[2024,8,27]],"date-time":"2024-08-27T13:01:50Z","timestamp":1724763710000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":3,"title":["Characterization of novel double-reporter strains of\n            <i>Mycobacterium abscessus<\/i>\n            for drug discovery: a study in mScarlet"],"prefix":"10.1128","volume":"12","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-9019-7334","authenticated-orcid":true,"given":"Clara M.","family":"Bento","sequence":"first","affiliation":[{"name":"i3S\u2014Instituto de Investiga\u00e7\u00e3o e Inova\u00e7\u00e3o e Sa\u00fade, Universidade do Porto, Porto, Portugal"},{"name":"IBMC\u2014Instituto de Biologia Celular e Molecular, Universidade do Porto, Porto, Portugal"},{"name":"Programa Doutoral em Biologia Molecular e Celular (MCBiology), Instituto de Ci\u00eancias Biom\u00e9dicas Abel Salazar da Universidade do Porto, Porto, Portugal"}]},{"given":"Kevin","family":"van Calster","sequence":"additional","affiliation":[{"name":"Laboratory for Microbiology, Parasitology and Hygiene (LMPH), Wilrijk, Belgium"}]},{"given":"Tatiana","family":"Piller","sequence":"additional","affiliation":[{"name":"Laboratory for Microbiology, Parasitology and Hygiene (LMPH), Wilrijk, Belgium"}]},{"given":"Gabriel S.","family":"Oliveira","sequence":"additional","affiliation":[{"name":"i3S\u2014Instituto de Investiga\u00e7\u00e3o e Inova\u00e7\u00e3o e Sa\u00fade, Universidade do Porto, Porto, Portugal"},{"name":"ICBAS\u2014Instituto de Ci\u00eancias Biom\u00e9dicas Abel Salazar da Universidade do Porto, Porto, Portugal"}]},{"given":"Linda","family":"de Vooght","sequence":"additional","affiliation":[{"name":"Laboratory for Microbiology, Parasitology and Hygiene (LMPH), Wilrijk, Belgium"}]},{"given":"Davie","family":"Cappoen","sequence":"additional","affiliation":[{"name":"Laboratory for Microbiology, Parasitology and Hygiene (LMPH), Wilrijk, Belgium"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4361-8911","authenticated-orcid":true,"given":"Paul","family":"Cos","sequence":"additional","affiliation":[{"name":"Laboratory for Microbiology, Parasitology and Hygiene (LMPH), Wilrijk, Belgium"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7657-2118","authenticated-orcid":true,"given":"M. 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