Antibiotic resistance is a critical global health concern, and antibiotic-tolerant cells further complicate treatment by contributing to infection relapses and promoting the emergence of resistant mutants. While tolerant cells can evolve into resistant populations, their phenotypic and genetic profiles remain poorly defined. In this study, we used Escherichia coli and the fluoroquinolone antibiotic ofloxacin to investigate the evolutionary dynamics of antibiotic-mediated adaptation. Through adaptive laboratory evolution (ALE) experiment, we found that tolerance and resistance can arise independently across different populations, despite being subjected to identical conditions, highlighting the unpredictability and diversity of bacterial evolutionary responses.
We assessed bacterial fitness by measuring lag phase duration, doubling time, competition score, redox activity, and intracellular ATP levels. Notably, no strong correlation was observed between these fitness parameters and the development of antibiotic tolerance or resistance. The whole genome sequencing of evolved strains revealed both shared and unique mutations. While some populations exhibited single-nucleotide polymorphisms (SNPs) in key metabolic genes like icd (involved in the citric acid cycle), the majority displayed highly diverse mutational profiles with no evidence of a conserved evolutionary pathway. Intriguingly, we also identified mutant strains with markedly lower minimum inhibitory concentrations (MICs) than the parental strain, yet with remarkably high tolerance to ofloxacin, revealing novel phenotypic traits.
Altogether, this work maps the complex phenotypic and genetic adaptations associated with fluoroquinolone treatment and provides new insights into the multifaceted nature of bacterial survival strategies. This study has been accepted for publication in mSystems, a journal of the American Society for Microbiology (ASM), and is available as a preprint on bioRxiv (1).
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