Unveiling the Impact of Extracellular Polymeric Substances (EPS) on the Conjugative Transfer of Antibiotic Resistance Genes (ARGs)

The spread of antibiotic resistance genes (ARGs) via horizontal gene transfer (HGT) in wastewater treatment processes presents a critical One-Health challenge. While extracellular polymeric substances (EPS) are known to envelop microbial cells and mediate intercellular interactions, their role in conjugation, the predominant HGT mode, remains unclear. Herein, we developed an in vivo framework to investigate the impacts of EPS on conjugation. Simulating the generation of antibiotic-resistant <i>Pseudomonas aeruginosa</i>, a critical ESKAPE pathogen, we found that EPS significantly shaped conjugative behaviors with their depletion consistently reducing conjugation occurrences. Mechanistic investigations revealed that while EPS removal increased the cell membrane permeability, community-level reactive oxygen species (ROS), and virulence gene expression, it also led to decreased intracellular energy production and diminished transcription of key conjugation components. Furthermore, EPS depletion compromised the physical integrity of microbial community structures such as biofilms, weakened cell-to-cell contact, and reduced biomass of microbes involved in conjugation. These factors collectively determine the fate of conjugation events. To further validate the regulatory role of EPS, we engineered a CRISPR-ddCas12a system to repress EPS biosynthesis, significantly suppressing the conjugation of ARGs. This work provides critical insights into conjugation mechanisms and underscores the potential of targeting EPS to limit conjugation in wastewater treatment.