The broad-spectrum antibiotic ciprofloxacin is frequently used to treat infections caused by a wide range of bacterial pathogens. We utilized a morbidostat device to evolve ciprofloxacin resistance in two clinically-relevant species of Gram-negative bacteria: Escherichia coli, Acinetobacter baumannii and Pseudomonas aeruginosa.

The broad-spectrum antibiotic ciprofloxacin is frequently used to treat infections caused by a wide range of bacterial pathogens. Unfortunately, the efficacy of this potent and well-tolerated drug is diminishing due to the rise and spread of microbial resistance. In this study, we utilized a morbidostat device to evolve ciprofloxacin resistance in three clinically-relevant species of Gram-negative bacteria: Escherichia coli, Acinetobacter baumannii and Pseudomonas aeruginosa. Our comparative resistomics approach, based on experimental evolution and deep sequencing of evolving microbial populations in time series, elucidated the dynamics and mechanisms of antibiotic resistance acquisition in these clinically-relevant pathogens. Disparate dosing regimens led to the emergence of numerous resistance-conferring mutations, including both clinically observed and uncommon genetic alterations. Along with observing the impact of varied dosing concentrations, the sample design permitted tracking of evolutionary trajectories, revealing a stepwise pattern of resistance mutation expansion. Taken together, the results of this study support the unique utility of a morbidostat design in antibiotic resistance research from basic science exploration to pre-clinical trial investigations of new drugs in development.