Adaptive Laboratory Evolution under gradual ciprofloxacin stress leads to efflux-mediated ciprofloxacin resistance and an increase in endogenous antibiotic production in Streptomyces coelicolor

Streptomyces are soil-dwelling bacteria harboring a vast reservoir of transcriptionally silent antibiotic-resistant genes. Mobilization of these resistance genes to pathogens results in the dissemination of multidrug resistance when exposed to differential antibiotic pressure. To determine the evolution of resistance, we employed adaptive laboratory evolution (ALE) in Streptomyces coelicolor under gradually increasing concentrations of ciprofloxacin, starting at the minimum inhibitory concentration (MIC) to 4X MIC. The evolved strain CipRr exhibited elevated resistance towards ciprofloxacin with cross-resistance to other fluoroquinolones, such as ofloxacin and moxifloxacin, and collateral sensitivity to the protein synthesis inhibitors chloramphenicol and tetracycline. Compared to the WT cells, CipRr showed elevated production of secondary metabolites actinorhodin and undecylprodigiosin, altered morphology, and reduced ROS production. Whole Genome Sequencing revealed mutations in transcriptional regulators and other metabolic genes, leading to enhanced activity of efflux pumps that conferred high-level ciprofloxacin resistance. Overall, this study demonstrates that evolutionary strategies in antibiotic producers such as streptomyces can help uncover the genetic determinants linked to antibiotic resistance, secondary metabolism, and morphological differentiation.