Revealing transcriptomic responses in Escherichia coli during early antibiotic exposure

The earliest responses of pathogenic bacteria to antibiotics can affect the outcome of an infection. While long-term adaptations have been extensively studied, the immediate transcriptional changes that unfold immediately following antibiotic exposure remain poorly understood. Here, we applied iModulon analysis to time-resolved transcriptomic data from Escherichia coli exposed to subinhibitory concentrations of two antibiotics (ampicillin and ciprofloxacin), capturing transcriptional regulatory changes occurring within the first 30 min of exposure. This analysis proposes an integrated, three-phase response model: an immediate and sustained primary response that broadly activates stress programs, a transient secondary response that restores redox balance, and a tertiary response that supports long-term survival through metabolic remodeling and antibiotic-specific defenses. These results highlight a coordinated and dynamic regulatory strategy describing how metabolic, redox, and stress responses are integrated to manage the physiological challenges of antibiotic stress. By disentangling these overlapping transcriptional regulatory programs, this work offers a genome-scale understanding of how early regulatory programs are engaged immediately after antibiotic exposure. Together, these findings provide a structured framework for characterizing complex transcriptomic responses and generating testable hypotheses about the regulatory logic that shapes the understudied early phase of antibiotic exposure.IMPORTANCEInitial bacterial responses to antibiotics are important for survival and can influence the development of tolerance and resistance. However, this period remains poorly understood, in part, because the transcriptional responses that unfold within minutes of antibiotic exposure are complex and difficult to interpret. In this study, we applied novel data generation and data analytics approaches to resolve the regulatory structure of the initial response of Escherichia coli to two antibiotics. We identify a three-phase process that explains how E. coli coordinates stress responses, maintains redox homeostasis, and initiates downstream protective programs. The novel transcriptomic analytics elucidate independently regulated sets of genes that constitute cellular processes. By identifying the regulatory modules that change over this initial timescale, we can deconvolute the response based on first principles of cellular physiology.