Background: Antibiotics at sub-inhibitory concentrations can rewire bacterial regulatory networks, impacting virulence. Objective: The way that exposure to selected antibiotics (ciprofloxacin, amikacin, azithromycin, ceftazidime, and meropenem) below their minimum inhibitory concentration (sub-MIC) modulates the physiology of Pseudomonas aeruginosa is examined in this study using growth-phase-resolved analysis. Methods: Standard P. aeruginosa strain cultures were exposed to ¼ and ½ MIC to determine the growth kinetics under antibiotic stress. The study measured protease and pyocyanin production and the expression level of important quorum sensing and virulence genes (lasI/R, rhlI/R, pqsR/A, and phzA) at different growth phases. Results: Meropenem produced the most noticeable growth suppression at ½ MIC. Sub-MIC antibiotics did not completely stop growth, but caused distinct, dose-dependent changes. Azithromycin eliminated protease activity in all phases and had a biphasic effect on pyocyanin. Ciprofloxacin consistently inhibited both pyocyanin and protease in all phases. The effects of amikacin varied by phase and dose, while β-lactams markedly increased pyocyanin production during the log phase. In contrast to the plateau phase, when expression was often downregulated or unchanged, most quorum-sensing- and virulence-associated genes showed significant upregulation during the death phase under sub-MIC exposure. Conclusions: These findings indicate that sub-MIC antibiotics act as biochemical signal modulators, preserving stress-adapted sub-populations that, in late growth phases, activate quorum sensing and stress tolerance pathways.
