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Beyond known resistance mechanisms, little is understood about the cellular responses that enable resistant\n                      <jats:italic toggle=\"yes\">E. coli<\/jats:italic>\n                      to grow under these antibiotic stresses. This study aimed to identify genes that support bacterial growth under SUL and TMP stress. Two saturated transposon mutant libraries were constructed in resistant\n                      <jats:italic toggle=\"yes\">E. coli<\/jats:italic>\n                      MG1655 harboring either\n                      <jats:italic toggle=\"yes\">dfrA1<\/jats:italic>\n                      or\n                      <jats:italic toggle=\"yes\">sul2<\/jats:italic>\n                      . They were grown with and without 1\/2 and 1\/4 minimum inhibitory concentration (MIC) of sulfamethoxazole (SMX) or TMP, and mutant depletion was assessed via transposon-directed insertion-site sequencing. At 1\/2 MIC, 36 and 89 genes were identified as conditionally essential during SMX and TMP exposure, while 5 and 2 genes were classified as conditionally essential at 1\/4 MIC. Genes identified as conditionally essential at 1\/4 MIC were also important at 1\/2 MIC. Conditionally essential genes belonged to lipopolysaccharide biosynthesis, peptidoglycan metabolism, energy production, membrane integrity, phosphate metabolism, and stress responses, highlighting the role of these factors in maintaining cell stability under SMX and TMP stress. Validation with 10 conditionally essential genes (\n                      <jats:italic toggle=\"yes\">apaH, mtn, surA, waaO, nlpI, prc, wzxE, fadR, degP,<\/jats:italic>\n                      and\n                      <jats:italic toggle=\"yes\">tpiA<\/jats:italic>\n                      ) showed that deletion mutants indeed exhibited growth defects and two- to eightfold reductions in MIC under antibiotic stresses compared to their parent strains. This study highlights cellular responses to SMX and TMP under antibiotic stress, and it has identified a list of genes whose products may serve as potential helper drug targets to resensitize resistant\n                      <jats:italic toggle=\"yes\">E. coli<\/jats:italic>\n                      to SMX and TMP treatments.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>IMPORTANCE<\/jats:title>\n                    <jats:p>\n                      Sulfonamides (SULs) and trimethoprim (TMP) are broad-spectrum antimicrobials. They are commonly used to treat infections in both humans and animals. Resistance against SUL and TMP is widespread in pathogenic bacteria, and there is a need to overcome this problem. One possibility is to target the cellular mechanism by which the resistant bacteria adapt to growth in the presence of the antimicrobials. In this study, we identify the genes, besides the resistance genes, which enable resistant\n                      <jats:italic toggle=\"yes\">Escherichia coli<\/jats:italic>\n                      to grow in the presence of SUL and TMP. We further show that knocking out many of these genes attenuates the resistant\n                      <jats:italic toggle=\"yes\">E. coli<\/jats:italic>\n                      for growth during SUL and\/or TMP stress, irrespective of which SUL- or TMP-resistant gene the bacteria carry. The gene products of these genes may serve as potential helper drug targets to resensitize resistant\n                      <jats:italic toggle=\"yes\">E. coli<\/jats:italic>\n                      to sulfamethoxazole and TMP treatments.\n                    <\/jats:p>\n                  <\/jats:sec>","DOI":"10.1128\/spectrum.00603-26","type":"journal-article","created":{"date-parts":[[2026,7,29]],"date-time":"2026-07-29T13:01:40Z","timestamp":1785330100000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":0,"title":["Genome-wide identification of conditionally essential genes for growth in the presence of sulfamethoxazole and trimethoprim in sulfamethoxazole- and trimethoprim-resistant\n                    <i>Escherichia coli<\/i>"],"prefix":"10.1128","volume":"14","author":[{"given":"Sajid","family":"Nisar","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/035b05819","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Veterinary and Animal Sciences, Faculty of Health and Medical Sciences, University of Copenhagen","place":["Frederiksberg, Denmark"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mosaed S.","family":"Alobaidallah","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/0149jvn88","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, King Saud bin Abdulaziz University for Health Sciences","place":["Jeddah, Saudi Arabia"]},{"id":[{"id":"https:\/\/ror.org\/009p8zv69","id-type":"ROR","asserted-by":"publisher"}],"name":"King Abdullah International Medical Research Center","place":["Jeddah, Saudi Arabia"]},{"id":[{"id":"https:\/\/ror.org\/02fkh1w66","id-type":"ROR","asserted-by":"publisher"}],"name":"Ministry of the National Guard\u2014Health Affairs","place":["Jeddah, Saudi Arabia"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sandra M.","family":"Wellner","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/035b05819","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Veterinary and Animal Sciences, Faculty of Health and Medical Sciences, University of Copenhagen","place":["Frederiksberg, Denmark"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nader","family":"Abdelmalek","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/01bnjbv91","id-type":"ROR","asserted-by":"publisher"}],"name":"Unit of Microbiology and Virology, Department of Biomedical Sciences, University Hospital of Sassari, University of Sassari","place":["Sassari, Italy"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mauro M.","family":"Saraiva","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/00987cb86","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Pathology, Reproduction, and One Health, Universidade Estadual Paulista-Unesp-FCAV","place":["Jaboticabal, Brazil"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5460-9178","authenticated-orcid":true,"given":"Line E.","family":"Thomsen","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/035b05819","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Veterinary and Animal Sciences, Faculty of Health and Medical Sciences, University of Copenhagen","place":["Frederiksberg, Denmark"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6225-6587","authenticated-orcid":false,"given":"John E.","family":"Olsen","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/035b05819","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Veterinary and Animal Sciences, Faculty of Health and Medical Sciences, University of Copenhagen","place":["Frederiksberg, Denmark"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"235","reference":[{"key":"e_1_3_4_2_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0140-6736(24)01867-1"},{"key":"e_1_3_4_3_2","unstructured":"O\u2019Neill J. 2016. 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