Genome-wide identification of conditionally essential genes for growth in the presence of sulfamethoxazole and trimethoprim in sulfamethoxazole- and trimethoprim-resistant Escherichia coli.
UNLABELLED: Resistance to sulfonamides (SULs) and trimethoprim (TMP) in Escherichia coli threatens their clinical relevance. Beyond known resistance mechanisms, little is understood about the cellular responses that enable resistant E. coli 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 E. coli MG1655 harboring either dfrA1 or sul2. 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 (apaH, mtn, surA, waaO, nlpI, prc, wzxE, fadR, degP, and tpiA) 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 E. coli to SMX and TMP treatments. IMPORTANCE: 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 Escherichia coli to grow in the presence of SUL and TMP. We further show that knocking out many of these genes attenuates the resistant E. coli 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 E. coli to sulfamethoxazole and TMP treatments.