Perfluoroalkyl sulfonate alternatives, such as 6:2 chlorinated polyfluorinated ether sulfonic acid (6:2 Cl-PFESA), are increasingly detected in wastewater treatment systems, yet their ecological risks remain poorly understood. Here, we used a multi-level analytical framework that integrated long-term exposure experiments, ARG and mobile genetic element profiling, EPS-associated extracellular DNA characterization, oxidative stress assays, and microbial community analysis to uncover mechanistic links between 6:2 Cl-PFESA exposure and ARG dissemination potential that could not be captured by previous studies focused solely on contaminant occurrence or ARG abundance. 6:2 Cl-PFESA treatment significantly promoted ARG proliferation, with tetracycline and sulfonamide resistance genes showing the strongest enrichment. Extracellular ARGs increased more sharply than intracellular ones, with the increases closely correlated with mobile genetic elements, such as intI1 , IS CR1 , and Tn 916 /Tn 1545 , suggesting increased genetic mobility potential rather than direct evidence of horizontal transfer. Concurrently, 6:2 Cl-PFESA treatment stimulated extracellular polymeric substance secretion and shifted extracellular polymeric substances composition toward protein-rich fractions, resulting in the immobilization of large amounts of extracellular DNA and the creation of reservoirs that stabilized the ARGs. At the cellular level, PFESA exposure triggered reactive oxygen species accumulation, oxidative stress responses, membrane disruption, and efflux pump activation, which facilitated DNA release and increased its availability for potential mobilization. Microbial community analysis revealed that PFESA exposure reshaped bacterial and archaeal consortia and selectively enriched resistant and syntrophic taxa (e.g., Dechloromonas , Geobacter , Syntrophobacter , Methanobacterium ), which acted as ARG/mobile genetic element hosts. Integrating these findings, we propose a multi-level mechanism whereby 6:2 Cl-PFESA exposure promotes ARG enrichment and enhances their dissemination potential through a combination of genetic, extracellular, cellular, and ecological pathways. This study advances the existing literature by identifying PFAS alternatives as previously overlooked drivers of ARG enrichment and dissemination potential during anaerobic digestion, thereby raising new biosafety concerns for sludge management and expanding current understanding of PFESA environmental risk. • Intra/extracellular ARGs increased during anaerobic digestion with 6:2 chlorinated polyfluorinated ether sulfonic acid (6:2 Cl-PFESA). • Extracellular ARGs showed enrichment and MGE links suggesting horizontal transfer. • PFESA exposure induced extracellular polymeric substances creation and DNA immobilization, creating ARG reservoirs. • DNA leakage, efflux pump activity, and plasmid transfer gene expression increased. • Microbial community reshaping favored ARG/MGE hosts, amplifying dissemination risks.
Li et al. (2026) studied this question.