Adaptive Resistance to Antibiotics in Escherichia coli

Abstract

Antimicrobial resistance (AMR) is expected to escalate into a major crisis as bacterial infections become increasingly harder to treat. Some difficult-to-eradicate bacteria may be treated with multiple antibiotic drugs. However, this can have unintended consequences, as exposure to drugs such as rifampicin can promote adaptive resistance in bacteria against other lethal antibiotics by inducing dormancy. Dormant cells have decreased adenosine triphosphate (ATP) levels, the energy source for numerous cellular processes. The reduced energy inhibits key processes targeted by the antibiotics, thereby enabling survival against antibiotic stress. ATP is synthesized with the aid of proton motive force (PMF) during respiration. Despite its significance as the central energy source, our understanding of the role of PMF in adaptive resistance is limited. In this work, we characterized the PMF in adaptive-resistant cells of Escherichia coli. These so-called dormant cells survived sequential exposure to rifampicin and ampicillin for several hours. Significantly, 1 out of 3 survivors remained highly motile. As motility is powered by the PMF, this suggested that the adapted population was not dormant. We systematically quantified cellular energetics at a single-cell level, finding that the clonal population bifurcated into high- and low-PMF subpopulations. Next, we measured the growth recovery after the removal of all antibiotics at a single-cell level. The high-PMF cells were able to recover and reproduce within 2 to 3 hours. This confirmed that the high-PMF cells were induced persisters. Our results indicate that persistence can arise without cellular dormancy. Variable PMF likely represents a bet-hedging strategy, where a fraction of the adapted cells retains a high PMF to escape the deleterious environment. Our comprehensive characterization of the chemotaxis, efflux pump activity, cellular respiration, and ATP levels further revealed that clonal populations employ multiple mechanisms to simultaneously adapt to antibiotic stresses.