Accelerated dissemination of antibiotic resistant genes via conjugative transfer driven by deficient denitrification...

Biofiltration systems harbored and disseminated antibiotic resistance genes (ARGs), when confronting antibiotic-contained wastewater. Biochar, a widely used environmental remediation material, can mitigate antibiotic stress on adjoining microbes by lowering the availability of sorbed antibiotics, and enhance the attachment of denitrifiers. Herein, bench-scale biofiltration systems, packed with commercial biochars, were established to explore the pivotal drivers affecting ARG emergence. Results showed that biofiltration columns, achieving higher TN removal and denitrification capacity, showed a significant decrease in ARG accumulation (p < 0.05). The relative abundance of ARGs (0.014 ± 0.0008) in the attached biofilms decreased to 1/5-folds of that in the control group (0.065 ± 0.004). Functional analysis indicated ARGs' accumulation was less attributed to ARG activation or horizontal gene transfer (HGT) driven by sorbed antibiotics. Most denitrifiers, like Bradyrhizobium, Geothrix, etc., were found to be enriched and host ARGs. Nitrosative stress from deficient denitrification was demonstrated to be the dominant driver for affecting ARG accumulation and dissemination. Metagenomic and metaproteomic analysis revealed that nitrosative stress promoted the conjugative HGT of ARGs mainly via increasing the transmembrane permeability and enhancing the amino acid transport and metabolism, such as cysteine, methionine, and valine metabolism. Overall, this study highlighted the risks of deficient denitrification in promoting ARG transfer and transmission in biofiltration systems and natural ecosystems.