Abstract
In most Earth environments, bacteria predominantly exist within surface-associated communities known as biofilms, where they are embedded in an extracellular matrix. These collective structures play a critical role in bacterial physiology and significantly shape their evolutionary trajectories, contributing to the development of antimicrobial resistance and enhancing bacterial resilience to treatments, with profound implications for public health. This study assessed the impact of the biofilm lifestyle on the emergence of resistance to gentamicin, an aminoglycoside antibiotic, in one laboratory reference strain and seven Escherichia coli isolates from food-processing environments. Throughout a one-month evolution experiment, we observed that certain strains showed a markedly higher emergence of gentamicin-resistant variants in biofilms than in planktonic states, with the emergence of stable variants being closely linked to biofilm maturation. Genomic and phenotypic analyses of gentamicin-resistant (GenR) variants uncovered varied adaptive strategies among the strains. GenR variants from two food-processing isolates (Ec709 and Ec478) displayed point mutations in genes associated with central carbon metabolism (aceE, ygfZ, …) and cell respiration (atpG, cydA, …), while retaining relative growth and colonization capacities. Conversely, GenR variants from the reference strain (Ec1655) adapted preferentially through large genomic deletions, including consistent loss of the peptide transporter gene sbmA, significantly altering cellular fitness. These findings highlight the complexity of adaptive evolution in biofilms and underscore the importance of investigating diverse strains to grasp the full spectrum of adaptation in natural bacterial populations.