The diverse phenotypic and mutational landscape induced by fluoroquinolone treatment

Antibiotic resistance remains a major public health challenge, yet the broader effects of antibiotic treatment on bacterial tolerance, resistance, and fitness are not fully understood. In this study, we investigated how Escherichia coli adapts to fluoroquinolone stress using adaptive laboratory evolution. Cell populations were subjected to repeated cycles of high-dose ofloxacin exposure followed by drug-free recovery, a dynamic model that imposes alternating selective pressure and inter-strain competition, reflecting real-world clinical or environmental conditions. Our results demonstrate that tolerance and resistance can evolve independently, even under identical conditions, leading to diverse phenotypic outcomes. Notably, we observed the emergence of mutants with high ofloxacin tolerance but reduced minimum inhibitory concentrations, an outcome that challenges conventional understanding. Fitness traits, including lag phase duration, doubling time, competition score, redox activity, and ATP levels, were variably affected across evolved strains, with no consistent correlation between fitness and tolerance or resistance. Whole-genome sequencing revealed both known and novel mutations, with limited convergence across populations. For example, while mutations in the icd gene were commonly observed, many other mutations, including in cyoE, lgoT, yghC, rnd, dld, and uidB, were unique to individual lineages. The lack of convergence across evolved populations may reflect the influence of competitive dynamics during recovery phases, where differing growth advantages shape selection in parallel with antibiotic pressure. These findings underscore the complexity of microbial adaptation and highlight how fluctuating environments and population-level interactions can drive non-uniform evolutionary outcomes.IMPORTANCEAntibiotic resistance poses a critical global health threat, with antibiotic-tolerant cells further complicating treatment by promoting infection relapse and enabling resistance mutations. Though tolerant cells can evolve into resistant strains, their phenotypic and genotypic characteristics are still poorly understood. In this study, we used adaptive laboratory evolution to generate several distinct ofloxacin-resistant mutants and examined their fitness (e.g., lag phase), metabolic traits (e.g., ATP levels), and genetic adaptations through whole-genome sequencing. We uncovered novel findings, including highly tolerant mutants exhibiting unexpectedly low minimum inhibitory concentrations and others with shorter lag phases, challenging conventional patterns in bacterial resistance evolution. Our findings provide critical insights into the diverse pathways and mechanisms underpinning bacterial adaptation, underscoring the complexity of resistance evolution.