Antibiotic exposure and recovery of persister states influence virulence phenotypes in Pseudomonas aeruginosa

Abstract The pathogenicity of P. aeruginosa is not only mediated by its resistance mechanisms but also by its ability to form persister cells. The persister state is a transient condition in which cells retain a dormant, non- or slow-growing state during exposure to stressors, such as high doses of bactericidal antibiotics, but can resume growth once the stress is removed. In this study, we investigated in vitro the virulence phenotype and stress-induced modulation of persisters in P. aeruginosa PA14 exposed to 30 × the minimum inhibitory concentration (MIC) of imipenem (60 µg/mL) or ciprofloxacin (3.75 µg/mL). Using a biphasic killing assay, we detected persisters with survival levels of 4.8 and 3.4 log 10 CFU/mL at 24 h for imipenem and ciprofloxacin, respectively. MIC assays confirmed the absence of acquired resistance. Flow cytometry revealed a time-dependent accumulation of cells with intermediate redox activity and membrane integrity, consistent with the persister phenotype. Both planktonic and biofilm-associated populations harbored persisters, with biofilms exhibiting a greater amount of tolerant cells. Furthermore, persister cells displayed reduced pigment production, biofilm formation, and phagocytosis survival. Gene expression analysis revealed upregulation of higB and pqsA in persisters, along with distinct profiles in recovered cells. Pre-exposure to hydrogen peroxide (H 2 O 2 ; 0.4%) increased survival by > 1 log 10 following antibiotic treatment. Additionally, pretreatment with 5-fluorouracil (5-FU; 256 µg/mL) increased imipenem persistence by twofold. Altogether, our findings show that oxidative and genotoxic stresses promote persistence, and that macrophage interactions modulate persister physiology, offering insight into host–pathogen dynamics and potential therapeutic avenues.