Divergent Evolutionary Trajectories of Pseudomonas aeruginosa PAO1 under Trace versus Preservative-Level Antimicrobial Methylisothiazolinone Exposure

Isothiazolinones are widely used nonantibiotic antimicrobials with high electrophilic reactivity toward bacterial protein thiols. Although this reactivity leads to rapid degradation and low environmental persistence, their potential to drive cryptic microbial evolution remains poorly understood. Here, we focused on methylisothiazolinone (MIT), a widely used isothiazolinone, and conducted a 60-cycle experimental evolution of Pseudomonas aeruginosa PAO1 across a concentration gradient spanning environmentally relevant (10 μg/L) to preservative-use (8-16 mg/L) levels. We demonstrate an exposure-level-dependent bifurcation in evolutionary strategies. Trace-level MIT exposure enhanced horizontal gene transfer capacity (from 0.0520 ± 0.0006 to 0.0764 ± 0.0008) through membrane remodeling, including elevated membrane potential, reduced extracellular polymeric substances, and 2.79-fold induction of indole signaling. In contrast, preservative-level MIT exposure drove key mutations (e.g., mexR deletion) and metabolic-transcriptional rewiring, increasing minimal inhibitory concentrations of Meropenem by 8- to 16-fold with minimal fitness costs. Furthermore, the 16 mg/L-evolved lineages exhibited hypervirulence, causing 100% mortality within 24 h in a Galleria mellonella model compared to 90% ancestral survival. These findings demonstrate that even trace exposure to highly bioactive antimicrobials can reshape microbial evolution and accelerate resistance emergence, highlighting unrecognized evolutionary risks and providing a critical scientific basis for refining their risk assessment and management frameworks.