Unraveling novel strategies of adaptation to antibiotics using low-drug selection

Abstract

Although antimicrobial resistance (AMR) poses a major biomedical challenge, early and often subtle adaptations that precede high-level resistance remain poorly understood. Current surveillance and diagnostic frameworks emphasize primarily on clinically relevant, high-level AMR. Bacteria are frequently exposed to sub-inhibitory levels of antibiotics in natural and clinical settings due to anthropogenic contamination, and unregulated antibiotic usage and their subsequent disposal, respectively. Consequently, subtle and low-level adaptation to antibiotics, i.e., precursors and drivers of resistance, are often overlooked and continue to remain under-explored. To address this gap, we employed laboratory evolution and exposed Escherichia coli to sub-inhibitory levels of antibiotics targeting diverse cellular processes. Through a combination of genetic, molecular, and physiological analyses, we identified relatively lesser-known modes of bacterial response to antibiotics and uncovered distinct adaptive strategies. We show that transient resistance to trimethoprim (an antifolate drug) is mediated via reversible amplification of folA, which codes for the drug target, dihydrofolate reductase. We also demonstrate adaptation to the ribosomal inhibitor, spectinomycin, without a change in the drug-MIC (minimum inhibitory concentration), where MIC is defined as the minimum concentration of an antibiotic required to inhibit bacterial growth. Our results establish that low-level adaptation to spectinomycin is, surprisingly, mediated by loss of an efflux pump via changes in the proton motive force sans classical, resistance-conferring mutations. We further show that resistance to beta-lactam antibiotics could be mediated by mutations in a sigma-factor (σ70). These findings emphasize the evolutionary significance of transient, plastic, and regulatory mechanisms in enabling bacterial survival in unfavourable environments. This work advances our understanding of the evolution of resistance within a ‘One Health’ framework, thereby underscoring the importance of targeting early adaptation to effectively mitigate the rise of resistance.