Antibiotic interactions and selection for resistance in biofilms

Abstract: The challenges posed by antibiotic-resistant bacteria in treating infections, particularly those associated with biofilms, require a deeper understanding of this lifestyle and its connection to resistance selection. Additionally, gaining insights into drug interactions is crucial for enhancing combination treatment efficacy and mitigating resistance development. This thesis is divided into these two main themes, each consisting of individual papers with specific objectives and aims that tackle these two themes.The first introduces a proof-of-concept microfluidic chip named Brimor, which demonstrates the selection of ciprofloxacin-resistant mutants in Escherichia coli biofilms at concentrations below the minimum inhibitory concentration (sub-MIC). Brimor exhibits potential applications beyond antibiotics and bacteria.The second explores the emergence of resistance in both planktonic and biofilm lifestyles. Using the FlexiPeg model and uropathogenic E. coli, the fitness cost and minimal selective concentrations were assessed for five antibiotics and six resistance-conferring mutations during biofilm and planktonic growth. This analysis revealed resistance development in both lifestyles at sub-MIC.Furthermore, an assay called CombiANT® was developed and validated with three major pathogens, enabling simple quantification and subsequent categorization of antibiotic interactions. This assay demonstrated comparable performance to the gold-standard checkerboard and time-kill assays. CombiANT® also shows potential for applications beyond antibiotics and bacteria.Isolate-specific interaction profiling was emphasized as crucial among five important Gram-negative pathogens for achieving precise and effective combination therapy. Interactions of clinically used antibiotic combinations varied significantly between and within susceptible species, with additive and antagonistic interactions being the most common. Only a small percentage exhibited clinically relevant synergy.The mutations associated with synergy and loss of synergy for the tetracycline and spectinomycin combination in E. coli was elucidated. Genetic changes associated with efflux regulation and metabolic pathways were identified as factors contributing to the loss of synergy in mutants. The bioavailability model was the prevailing mechanism of action accounting for synergy and loss of synergy for the combination.In summary, the papers presented in this thesis provide valuable insights on antibiotic resistance selection in biofilms, antibiotic interactions, and the development of innovative tools for studying biofilms and combination therapies. Further understanding of these factors is necessary for applying these findings in clinical settings and to optimize combination strategies for effective personalized therapy and antibiotic stewardship.