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Institute of Ecology (NIOO-KNAW)https://www.ebi.ac.uk/ena/browser/view/PRJNA844400Escherichia coli str. K-12 substr. MG1655We utilised direct competitive fitness measures among each mutant with its three single-mutation neighbours to describe the fitness landscape of a network of three mutations in a beta-lactamase enzyme (TEM) at sub-lethal concentrations of the antibiotic cefotaxime in a structured and unstructured environment. We deep sequenced populations to determine the frequency of different alleles at different time points. Custom barcodes were introduced during PCR amplification of the samples, so that multiple populations - comprising different sets of genotype within the fitness landscape - could be pooled for each Nextera XT library preparation. The different sets of genotypes for which competitions were performed are comprised of each genotype in the landscape, with its three Hamming distance = 1 neighbours. In addition, a bulk competition with all eight genotypes in the landscape was performed. For an overview of the samples, libraries, barcodes and additional methods, please consult the manuscript and supplementary materials. The sample names indicate the replicate (1-4), the medium (agar or liquid), the time post inoculation of sample collection in hours (0, 24 or 48 h), and finally the initial antibiotic concentration in the medium (0, 0.02 and 0.04 ug / mL cefotaxime). As the same initial populations were used for competitions in liquid and agar media, only a timepoint is indicated for T = 0. The exception is replicate 4, were different starting populations were used as indicated by the sample names.pathogen:bacteriumpathogen:priorityxref:EuropePMC:PMC10294655pathogenbacteriaENAfalseEscherichia coli str. K-12 substr. MG1655Escherichia coli str. K-12 substr. MG1655 Raw sequence reads2025-09-242022-06-03PRJNA844400511145