{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,6]],"date-time":"2026-10-06T02:32:48Z","timestamp":1791253968826,"version":"4.1.0"},"reference-count":38,"publisher":"American Society for Microbiology","issue":"6","license":[{"start":{"date-parts":[[2026,6,23]],"date-time":"2026-06-23T00:00:00Z","timestamp":1782172800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2026,6,23]],"date-time":"2026-06-23T00:00:00Z","timestamp":1782172800000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.asm.org\/non-commercial-tdm-license"}],"funder":[{"DOI":"10.13039\/501100007155","name":"Medical Research Council","doi-asserted-by":"publisher","award":["CC2081"],"award-info":[{"award-number":["CC2081"]}],"id":[{"id":"10.13039\/501100007155","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100010269","name":"Wellcome Trust","doi-asserted-by":"publisher","award":["CC2081"],"award-info":[{"award-number":["CC2081"]}],"id":[{"id":"10.13039\/100010269","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100000289","name":"Cancer Research UK","doi-asserted-by":"publisher","award":["CC2081"],"award-info":[{"award-number":["CC2081"]}],"id":[{"id":"10.13039\/501100000289","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["journals.asm.org"],"crossmark-restriction":true},"short-container-title":["mSystems"],"published-print":{"date-parts":[[2026,6,23]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:sec>\n                    <jats:title\/>\n                    <jats:p>\n                      Despite the importance of monitoring\n                      <jats:italic toggle=\"yes\">Mycobacterium tuberculosis<\/jats:italic>\n                      (Mtb) replication and antibiotic responses at the single-cell level, available tools to accurately monitor single Mtb replication are limited. Here, we report the development of Mtb-Timer, a fluorescent reporter in Mtb that exploits the intrinsic green-to-red photoconversion of the Timer protein as a proxy for Mtb replication. We generated an Mtb H37Rv strain expressing Timer (Timer\n                      <jats:sup>Mtb<\/jats:sup>\n                      ) and monitored\n                      <jats:italic toggle=\"yes\">in vitro<\/jats:italic>\n                      Mtb replication at the single-cell level by imaging and flow cytometry. Moreover, we show that exposure to the first-line anti-tuberculosis antibiotics isoniazid, rifampicin, ethambutol, and pyrazinamide resulted in a dose-dependent green-to-red photoconversion by imaging and flow cytometry. When we tested Mtb-Timer\n                      <jats:italic toggle=\"yes\">in cellulo<\/jats:italic>\n                      using primary human monocyte-derived macrophages, we observed that intracellular Mtb populations displayed a striking phenotypic heterogeneity. Single-cell Mtb-Timer green-to-red ratios analyzed by both fixed and live imaging revealed intracellular Mtb populations with high green-to-red ratios. In contrast, treatment with antibiotics showed a decrease in green-to-red ratios in a dose-dependent manner by imaging and flow cytometry. Collectively, these results establish Mtb-Timer as a robust tool for monitoring Mtb replication and antibiotic responses, both\n                      <jats:italic toggle=\"yes\">in vitro<\/jats:italic>\n                      and\n                      <jats:italic toggle=\"yes\">in cellulo<\/jats:italic>\n                      , by imaging and flow cytometry.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>IMPORTANCE<\/jats:title>\n                    <jats:p>\n                      Tuberculosis treatment is challenging in part because\n                      <jats:italic toggle=\"yes\">Mycobacterium tuberculosis<\/jats:italic>\n                      (Mtb) populations are phenotypically heterogeneous. Within the same infection, bacterial cells can exist in different physiological states, from actively replicating to slow-growing or non-replicating forms. To better study these processes, we developed and validated Mtb-Timer, a fluorescent reporter that enables visualization of bacterial growth dynamics. The system is based on a fluorescent protein that changes color over time: newly synthesized protein emits green fluorescence (GFP) and gradually shifts to red (DsRed) as it matures. The green-to-red fluorescence ratio, therefore, reflects bacterial replication activity. Using microscopy and flow cytometry in both\n                      <jats:italic toggle=\"yes\">in vitro<\/jats:italic>\n                      cultures and infected macrophages (\n                      <jats:italic toggle=\"yes\">in cellulo<\/jats:italic>\n                      ), Mtb-Timer enables the quantification of distinct bacterial subpopulations and the real-time monitoring of the green-to-red fluorescence ratio and bacterial burden during treatment with first-line antibiotics.\n                    <\/jats:p>\n                  <\/jats:sec>","DOI":"10.1128\/msystems.01796-25","type":"journal-article","created":{"date-parts":[[2026,5,19]],"date-time":"2026-05-19T12:55:18Z","timestamp":1779195318000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":3,"title":["Mtb-Timer: a fluorescent reporter to visualize\n                    <i>Mycobacterium tuberculosis<\/i>\n                    replication and antibiotic responses"],"prefix":"10.1128","volume":"11","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-3161-9984","authenticated-orcid":false,"given":"V\u00edctor","family":"Campo-P\u00e9rez","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/04tnbqb63","id-type":"ROR","asserted-by":"publisher"}],"name":"Host-Pathogen Interactions in Tuberculosis Laboratory, The Francis Crick Institute","place":["London, United Kingdom"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5155-2257","authenticated-orcid":false,"given":"Chak Hon","family":"Luk","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/04tnbqb63","id-type":"ROR","asserted-by":"publisher"}],"name":"Host-Pathogen Interactions in Tuberculosis Laboratory, The Francis Crick Institute","place":["London, United Kingdom"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Laure","family":"Botella","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/04tnbqb63","id-type":"ROR","asserted-by":"publisher"}],"name":"Host-Pathogen Interactions in Tuberculosis Laboratory, The Francis Crick Institute","place":["London, United Kingdom"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Julien","family":"Vaubourgeix","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/01ahyrz84","id-type":"ROR","asserted-by":"publisher"}],"name":"Institut de Recherche en Sant\u00e9 Digestive, Universit\u00e9 de Toulouse, INSERM, INRAE, ENVT, UPS","place":["Toulouse, France"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3199-0337","authenticated-orcid":true,"given":"Maximiliano G.","family":"Gutierrez","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/04tnbqb63","id-type":"ROR","asserted-by":"publisher"}],"name":"Host-Pathogen Interactions in Tuberculosis Laboratory, The Francis Crick Institute","place":["London, United 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