{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,21]],"date-time":"2026-10-21T22:29:14Z","timestamp":1792621754901,"version":"4.5.0"},"reference-count":28,"publisher":"American Society for Microbiology","issue":"3","license":[{"start":{"date-parts":[[2018,5,29]],"date-time":"2018-05-29T00:00:00Z","timestamp":1527552000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2018,7,5]],"date-time":"2018-07-05T00:00:00Z","timestamp":1530748800000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.asm.org\/non-commercial-tdm-license"}],"funder":[{"DOI":"10.13039\/100000060","name":"HHS | NIH | National Institute of Allergy and Infectious Diseases","doi-asserted-by":"publisher","award":["AI26170"],"award-info":[{"award-number":["AI26170"]}],"id":[{"id":"10.13039\/100000060","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000060","name":"HHS | NIH | National Institute of Allergy and Infectious Diseases","doi-asserted-by":"publisher","award":["U19AI111276"],"award-info":[{"award-number":["U19AI111276"]}],"id":[{"id":"10.13039\/100000060","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100003442","name":"Stony Wold-Herbert Fund","doi-asserted-by":"publisher","id":[{"id":"10.13039\/100003442","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100008881","name":"Potts Family Foundation","doi-asserted-by":"publisher","id":[{"id":"10.13039\/100008881","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["journals.asm.org"],"crossmark-restriction":true},"short-container-title":["mBio"],"published-print":{"date-parts":[[2018,7,5]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:p>\n                    Multidrug-resistant (MDR) tuberculosis, defined as tuberculosis resistant to the two first-line drugs isoniazid and rifampin, poses a serious problem for global tuberculosis control strategies. Lack of a safe and convenient model organism hampers progress in combating the spread of MDR strains of\n                    <jats:named-content content-type=\"genus-species\">Mycobacterium tuberculosis<\/jats:named-content>\n                    . We reasoned that auxotrophic MDR mutants of\n                    <jats:italic>M. tuberculosis<\/jats:italic>\n                    would provide a safe means for studying MDR\n                    <jats:italic>M. tuberculosis<\/jats:italic>\n                    without the need for a biosafety level 3 (BSL3) laboratory. Two different sets of triple auxotrophic mutants of\n                    <jats:italic>M. tuberculosis<\/jats:italic>\n                    were generated, which were auxotrophic for the nutrients leucine, pantothenate, and arginine or for leucine, pantothenate, and methionine. These triple auxotrophic strains retained their acid-fastness, their ability to generate both a drug persistence phenotype and drug-resistant mutants, and their susceptibility to plaque-forming mycobacterial phages. MDR triple auxotrophic mutants were obtained in a two-step fashion, selecting first for solely isoniazid-resistant or rifampin-resistant mutants. Interestingly, selection for isoniazid-resistant mutants of the methionine auxotroph generated isolates with single point mutations in\n                    <jats:italic>katG<\/jats:italic>\n                    , which encodes an isoniazid-activating enzyme, whereas similar selection using the arginine auxotroph yielded isoniazid-resistant mutants with large deletions in the chromosomal region containing\n                    <jats:italic>katG<\/jats:italic>\n                    . These\n                    <jats:italic>M. tuberculosis<\/jats:italic>\n                    MDR strains were readily sterilized by second-line tuberculosis drugs and failed to kill immunocompromised mice. These strains provide attractive candidates for\n                    <jats:italic>M. tuberculosis<\/jats:italic>\n                    biology studies and drug screening outside the BSL3 facility.\n                  <\/jats:p>\n                  <jats:p>\n                    <jats:bold>IMPORTANCE<\/jats:bold>\n                    Elimination of\n                    <jats:named-content content-type=\"genus-species\">Mycobacterium tuberculosis<\/jats:named-content>\n                    , the bacterium causing tuberculosis, requires enhanced understanding of its biology in order to identify new drugs against drug-susceptible and drug-resistant\n                    <jats:italic>M. tuberculosis<\/jats:italic>\n                    as well as uncovering novel pathways that lead to\n                    <jats:italic>M. tuberculosis<\/jats:italic>\n                    death. To circumvent the need for a biosafety level 3 (BSL3) laboratory when conducting research on\n                    <jats:italic>M. tuberculosis<\/jats:italic>\n                    , we have generated drug-susceptible and drug-resistant triple auxotrophic strains of\n                    <jats:italic>M. tuberculosis<\/jats:italic>\n                    suitable for use in a BSL2 laboratory. These strains originate from a double auxotrophic\n                    <jats:italic>M. tuberculosis<\/jats:italic>\n                    strain, H37Rv \u0394\n                    <jats:italic>panCD<\/jats:italic>\n                    \u0394\n                    <jats:italic>leuCD<\/jats:italic>\n                    , which was reclassified as a BSL2 strain based on its lack of lethality in immunocompromised and immunocompetent mice. A third auxotrophy (methionine or arginine) was introduced via deletion of\n                    <jats:italic>metA<\/jats:italic>\n                    or\n                    <jats:italic>argB<\/jats:italic>\n                    , respectively, since\n                    <jats:italic>M. tuberculosis<\/jats:italic>\n                    \u0394\n                    <jats:italic>metA<\/jats:italic>\n                    and\n                    <jats:italic>M. tuberculosis<\/jats:italic>\n                    \u0394\n                    <jats:italic>argB<\/jats:italic>\n                    are unable to survive amino acid auxotrophy and infect their host. The resulting triple auxotrophic\n                    <jats:italic>M. tuberculosis<\/jats:italic>\n                    strains retained characteristics of\n                    <jats:italic>M. tuberculosis<\/jats:italic>\n                    relevant for most types of investigations.\n                  <\/jats:p>","DOI":"10.1128\/mbio.00938-18","type":"journal-article","created":{"date-parts":[[2018,5,28]],"date-time":"2018-05-28T05:35:37Z","timestamp":1527485737000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":59,"title":["Rational Design of Biosafety Level 2-Approved, Multidrug-Resistant Strains of Mycobacterium tuberculosis through Nutrient Auxotrophy"],"prefix":"10.1128","volume":"9","author":[{"given":"Catherine","family":"Vilch\u00e8ze","sequence":"first","affiliation":[{"name":"Howard Hughes Medical Institute, Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jacqueline","family":"Copeland","sequence":"additional","affiliation":[{"name":"Howard Hughes Medical Institute, Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tracy L.","family":"Keiser","sequence":"additional","affiliation":[{"name":"Howard Hughes Medical Institute, Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Torin","family":"Weisbrod","sequence":"additional","affiliation":[{"name":"Howard Hughes Medical Institute, Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jacqueline","family":"Washington","sequence":"additional","affiliation":[{"name":"Department of Biology and Chemistry, Nyack College, Nyack, New York, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Paras","family":"Jain","sequence":"additional","affiliation":[{"name":"Howard Hughes Medical Institute, Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Adel","family":"Malek","sequence":"additional","affiliation":[{"name":"Howard Hughes Medical Institute, Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Brian","family":"Weinrick","sequence":"additional","affiliation":[{"name":"Howard Hughes Medical Institute, Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"suffix":"Jr.","given":"William R.","family":"Jacobs","sequence":"additional","affiliation":[{"name":"Howard Hughes Medical Institute, Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"235","reference":[{"key":"e_1_3_2_2_2","unstructured":"World Health Organization. 2017. Multidrug-resistant tuberculosis (MDR-TB). 2017 update. 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Preventive vaccination against tuberculosis with BCG. 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