Xenobiotic Stress-Driven Adaptation and Antibiotic Cross-Tolerance in Bacteria: Insights from Experimental Evolution and Multi-Omics Analyses

Abstract

Antibiotic resistance has become one of the most urgent health crises of the twenty-first century, with resistant infections now responsible for millions of deaths. Understanding how and why bacteria develop resistance, and what determines the rate at which they do, is not only important but also urgent. Bacterial exposure to diverse chemical stressors, including environmental xenobiotics and antibiotics, can significantly reshape cellular physiology and influence the evolution of antibiotic resistance. While antibiotic-driven resistance has been extensively studied, growing evidence suggests that non-antibiotic xenobiotics can modulate bacterial physiology and influence bacterial adaptation. However, the extent to which such exposures drive cross-resistance and reshape cellular pathways remains poorly understood. This thesis investigates how xenobiotics affect bacterial physiology and tolerance phenotypes using experimental evolution, phenotypic characterization and LC-MS/MS-based untargeted metabolomics and proteomics in Escherichia coli K-12 with a focus on identifying shared molecular and phenotypic responses associated with xenobiotic and antibiotic exposure. Chapter 2 presents a perspective review that establishes the conceptual framework of this thesis. Here, I synthesize current knowledge on xenobiotic-driven bacterial adaptations, integrating mechanistic insights, including alterations in membrane permeability, shared efflux responses, and global stress regulatory pathways. The broader ecological impact includes co-exposure scenarios and shifts in microbial community composition. This chapter highlights the need to move beyond antibiotic-centric models and proposes that xenobiotics represent a significant and less explored selective pressure in microbial ecosystems. Chapter 3 presents the investigation of the adaptation of E. coli K-12 under xenobiotic exposure using laboratory evolution combined with proteomics, metabolomics and phenotypic assays. The results reveal coordinated changes in metabolic pathways, membrane remodeling and redox homeostasis. The results also reveal that cellular responses extend beyond direct target pathways and contribute to cross tolerance to other compounds. Our data shows that even in cases where phenotypic resistance is limited, significant molecular reprogramming occurs, suggesting early-stage adaptation and initial cellular effects. This chapter also examines how exposure to a target specific antibiotic Fosfomycin can induce broader cellular responses beyond its primary mode of action. Chapter 4 explores bacterial evolution under multiple classes of antibiotics over 16 days, examining whether resistance evolution rates vary across compounds. It will further explore whether the breadth of drug-protein interactions may explain variation in resistance evolution. At this stage, the analysis focuses primarily on evolutionary outcomes. The integration with proteome-wide analyses to investigate off-target effects remains part of ongoing and future work. This combined framework will contribute to understanding how off-target effects of antibiotics may influence resistance evolution with potential implications for the development of effective combination therapies. Together, this thesis demonstrates that bacterial adaptation to xenobiotics is driven by interconnected physiological pathways rather than isolated molecular targets. This work supports the concept that xenobiotic exposure can contribute to increased antibiotic tolerance. By integrating conceptual and experimental approaches, this thesis provides mechanistic insights into the relationship between xenobiotic adaptation, antibiotic tolerance and resistance evolution. Collectively, these findings emphasize the importance of considering chemical complexity in resistance research and identify future directions for investigating the emergence and spread of antimicrobial resistance in both environmental and clinical settings