Metagenomic insights into suppressing antibiotic-resistant bacteria in mesocosm-scale constructed wetlands: calamus-biochar alleviates selective pressure and disrupts genetic co-occurrence network.

As a mainstream technology for the advanced treatment of wastewater treatment plant effluents, constructed wetlands (CWs) exhibit limited efficiency in antibiotic removal and may instead serve as reservoirs for antibiotic resistance genes (ARGs) and antibiotic-resistant bacteria (ARB). To address this, a mesocosm-scale CW amended with 4.0% calamus-biochar (PBC) filler was developed, achieving average antibiotic removal efficiencies above 92.7%. Compared with the blank system, the average ARGs removal efficiency increased by 54.2% and the proliferation of ARB was suppressed by an average of 65.1%. Furthermore, we found that the PBC filler adsorbed 41.8% of antibiotics while associating with only 1.4% of total culturable microorganisms, which may alleviate antibiotic selection pressure. Metagenomic analysis revealed that PBC filler reduced the normalized abundance (copies per cell) of mobile genetic elements (MGEs) by 0.68-5.98 cpc, accounting for 24.7-56.1%, weakened ARG-MGE co-occurrence and decreased the abundance of ARG-MGE co-localized contigs. Metagenome-assembled genome (MAG) analysis identified that Pseudomonadota was the dominant ARB phylum, predominantly harboring multidrug resistance genes and transposases, with a 56.7% reduction in relative abundance compared to the blank system. Batch experiments further confirmed that the PBC filler inhibited the potential for horizontal gene transfer (HGT) by sequestering ARGs. This study developed a CW system supplied with PBC filler for efficient removal of antibiotics, ARGs and ARB. It further elucidated the underlying mechanisms, with the PBC filler potentially decreasing antibiotic bioavailability and the potential for HGT of ARGs, thereby suppressing ARB proliferation.