Why Antimicrobial Resistance Does Not Perpetually Expand in an Antibiotics-Free Environment: Insight from Quorum Sensing

The persistence of antimicrobial resistance (AMR) in environmental reservoirs, even in the absence of direct antibiotic selection pressure, represents a critical challenge for global health. However, the mechanisms that prevent the unlimited expansion of environmental AMR remain poorly understood. In this study, we explore how quorum sensing (QS) plays a central role in constraining AMR under antibiotic-free conditions. Through long-term evolution experiments with antibiotic-resistant Pseudomonas aeruginosa strains ( P AMP and P CIP ), we observed the spontaneous emergence and accumulation of las R-deficient mutants (Δ las R), which act as social cheaters. These mutants exhibited a 2.44- to 4.88-fold reduction in minimum inhibitory concentration and impaired biofilm formation, leading to a 70–87.5% decrease in biofilm-protected survival against antibiotics. Moreover, the Δ las R frequency was strongly correlated with the decline in population-level resistance. Importantly, disruption of QS-mediated policing via reactive oxygen species scavengers accelerated the loss of resistance, confirming the role of las R in maintaining AMR. Furthermore, Δ las R possessed a growth advantage only in the absence of antibiotics, thus illustrating an evolutionary trade-off that naturally selects against resistance traits when selection pressure is removed. These findings provide a mechanistic explanation for the reversibility of environmental AMR and may aid the development of strategies targeting QS policing to mitigate antibiotic resistance.