From contaminant to accelerator: 6:2 Cl-PFESA reshapes microbial communities and exacerbates hidden antibiotic resistance threats

Perfluoroalkyl and polyfluoroalkyl substances (PFAS) are persistent environmental pollutants, and their phased-out legacy forms (e.g., PFOS) have been replaced by structural alternatives such as 6:2 chlorinated polyfluorinated ether sulfonic acid (6:2 Cl-PFESA). This compound is now widely detected in wastewater treatment systems, yet its ecological risks remain poorly understood. In a new research article published in Water & Ecology, a research team led by Xuening Zhang from Beijing Forestry University and Yilu Sun from Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, reveals that PFESA exposure significantly exacerbates antibiotic resistance gene (ARG) proliferation through multiple interconnected mechanisms, raising critical concerns about the biosafety of sludge management and the environmental risks of PFAS alternatives.

The study demonstrates that both extracellular ARGs (se-ARGs) and intracellular ARGs (si-ARGs) exhibited concentration-responsive increases during anaerobic digestion, with tetracycline and sulfonamide resistance genes showing the strongest enrichment. As the authors explain, "extracellular ARGs increased more sharply than intracellular ones, with the increases closely correlated with mobile genetic elements, such as intI1, ISCR1, and Tn916/1545, suggesting increased genetic mobility potential rather than direct evidence of horizontal transfer."

A key finding involves the restructuring of extracellular polymeric substances (EPS), which function as critical reservoirs for ARG persistence. PFESA exposure stimulated EPS secretion and shifted composition toward protein-rich fractions, resulting in the immobilization of large amounts of extracellular DNA. The study found that EPS-associated DNA accounted for 4.9% ~ 10.5% of the total DNA, which was 2~3 orders of magnitude higher than cell-free DNA content. This EPS-mediated retention creates stable reservoirs that prolong environmental residence time and increase genetic accessibility for potential microbial uptake.

At the cellular level, PFESA exposure triggered reactive oxygen species accumulation, oxidative stress responses, membrane disruption, and efflux pump activation. The research documented upregulation of stress-response genes, including SOS response genes (recA, lexA, sulA) and oxidative stress defense genes (soxS, soxR, oxyR). Concurrently, the team observed elevated membrane permeability indicators, such as alkaline phosphatase activity and malondialdehyde levels. These cellular perturbations facilitated DNA release into extracellular environments while simultaneously activating regulatory systems associated with genetic mobility.

Microbial community analysis revealed that PFESA exposure reshaped bacterial and archaeal consortia, selectively enriching resistant and syntrophic taxa including Dechloromonas, Geobacter, Syntrophobacter, and Methanobacterium. These organisms serve as potential ARG and mobile genetic element hosts, expanding ecological reservoirs that could support resistance dissemination. The persistence of these patterns across multiple operational cycles indicated long-term selective pressure rather than transient stimulation effects.

"The potential transfer of ARGs among unrelated bacterial taxa found in WWTPs is now recognized as an increasing human health risk," point out the authors. "However, similar studies on emerging PFAS alternatives such as 6:2 Cl-PFESA are lacking, so the possibility that these chemicals also exacerbate ARG transfer risks in anaerobic systems like WWTPs still requires systematic investigation."

This research provides mechanistic support for incorporating antimicrobial resistance indicators into environmental risk assessments of persistent pollutants, highlighting that PFAS alternatives may magnify resistance risks through EPS restructuring, cellular stress responses, and community reshaping rather than passive selection alone.

###

Contact the author:

Xuening Zhang

-Beijing Key Laboratory for Source Control Technology of Water Pollution, College of Environmental Science and Engineering, Beijing Forestry University, Beijing 100083, China

Zhangxn2025@bjfu.edu.cn

Yilu Sun

-Key Laboratory of Environmental Biotechnology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China

-University of Chinese Academy of Sciences, Beijing 100049, China

ylsun@rcees.ac.cn

The publisher KeAi was established by Elsevier and China Science Publishing & Media Ltd to unfold quality research globally. In 2013, our focus shifted to open access publishing. We now proudly publish more than 200 world-class, open access, English language journals, spanning all scientific disciplines. Many of these are titles we publish in partnership with prestigious societies and academic institutions, such as the National Natural Science Foundation of China (NSFC).