{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,23]],"date-time":"2026-10-23T20:06:06Z","timestamp":1792785966027,"version":"4.5.0"},"reference-count":25,"publisher":"Oxford University Press (OUP)","issue":"5","license":[{"start":{"date-parts":[[2026,9,30]],"date-time":"2026-09-30T00:00:00Z","timestamp":1790726400000},"content-version":"vor","delay-in-days":29,"URL":"https:\/\/creativecommons.org\/licenses\/by-nc\/4.0\/"}],"funder":[{"name":"Guangzhou National Laboratory","award":["GZNL2024A01009"],"award-info":[{"award-number":["GZNL2024A01009"]}],"id":[{"id":"https:\/\/ror.org\/03ybmxt82","id-type":"ROR","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2026,9,1]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:sec>\n                    <jats:title>Objectives<\/jats:title>\n                    <jats:p>para-Aminosalicylic acid is an antifolate prodrug activated in the folate biosynthesis pathway of Mycobacterium tuberculosis (Mtb), ultimately inhibiting dihydrofolate reductase encoded by dfrA. Mutations in dfrA coding and upstream regions have not been thoroughly investigated. This study aimed to assess mutations in dfrA and its upstream regulatory region in clinical Mtb isolates.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Methods<\/jats:title>\n                    <jats:p>We retrieved and analysed 6547 whole-genome sequences of Mtb clinical isolates. We identified 29 SNPs in the dfrA coding region and eight mutations in the thyA\u2013dfrA upstream region. The most prevalent coding mutations and upstream mutations were selected for functional evaluation using thermal stability analysis, GFP reporter assays and para-aminosalicylic acid drug susceptibility test (DST).<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>The coding mutations Trp142\u2192Gly, Gln28\u2192Leu and Arg32\u2192Pro were analysed for protein thermal stability and para-aminosalicylic acid susceptibility. Gln28\u2192Leu showed increased thermal stability relative to the WT, whereas Trp142\u2192Gly and Arg32\u2192Pro exhibited reduced stability. Consistently, para-aminosalicylic acid DST demonstrated that Gln28\u2192Leu conferred an 8-fold increase in para-aminosalicylic acid resistance, while Trp142\u2192Gly and Arg32\u2192Pro remained phenotypically susceptible. Moreover, GFP reporter assays revealed that G70T, G59T and C48A increased promoter activity by 13-fold, 4-fold and 3-fold, respectively, whereas the remaining mutations showed no significant effect. Findings were verified by para-aminosalicylic acid DST, where G70T, G59T and C48A showed para-aminosalicylic acid resistance compared with WT.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusions<\/jats:title>\n                    <jats:p>By integrating genomic analysis with functional assays of thermal stability, promoter activity and drug susceptibility, this study demonstrated that mutations in dfrA coding and upstream regulatory regions contribute to para-aminosalicylic acid resistance in Mtb clinical isolates.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.1093\/jacamr\/dlag211","type":"journal-article","created":{"date-parts":[[2026,9,9]],"date-time":"2026-09-09T11:51:20Z","timestamp":1788954680000},"source":"Crossref","is-referenced-by-count":0,"title":["Mutations in\n                    <i>dfrA<\/i>\n                    and its upstream region are associated with\n                    <i>para<\/i>\n                    -aminosalicylic acid resistance in\n                    <i>Mycobacterium tuberculosis<\/i>"],"prefix":"10.1093","volume":"8","author":[{"given":"Rong","family":"Zeng","sequence":"first","affiliation":[{"name":"Chest Hospital of Guangxi Zhuang Autonomous Region Department of Laboratory Medicine, , Liuzhou, Guangxi, 545005 ,","place":["China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Najeeb","family":"Ullah","sequence":"additional","affiliation":[{"name":"Wuhan Institute of Virology, Chinese Academy of Sciences , Wuhan 430071 ,","place":["China"]},{"name":"University of Chinese Academy of Sciences , Beijing 100049 ,","place":["China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jing","family":"Gu","sequence":"additional","affiliation":[{"name":"Wuhan Institute of Virology, Chinese Academy of Sciences , Wuhan 430071 ,","place":["China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jiao-Yu","family":"Deng","sequence":"additional","affiliation":[{"name":"Wuhan Institute of Virology, Chinese Academy of Sciences , Wuhan 430071 ,","place":["China"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"286","published-online":{"date-parts":[[2026,9,30]]},"reference":[{"key":"2026093004414944300_dlag211-B1","author":"World Health Organization"},{"key":"2026093004414944300_dlag211-B2","doi-asserted-by":"publisher","first-page":"617","DOI":"10.1038\/s41579-024-01025-1","article-title":"Drug-resistant tuberculosis: a persistent global health concern","volume":"22","author":"Farhat","year":"2024","journal-title":"Nat Rev Microbiol"},{"key":"2026093004414944300_dlag211-B3","doi-asserted-by":"publisher","first-page":"132","DOI":"10.1056\/NEJM195201242460404","article-title":"Effect of combined therapy (dihydrostreptomycin and PAS) on the emergence of streptomycin-resistant strains 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