Conjugation effects on antibiotic resistance genes at various salt Levels: Insights from single-factor and simulation study

The dissemination mechanisms of antibiotic resistance genes (ARGs) under salinity fluctuations remain poorly understood, despite their critical implications for environmental resistance ecology. This study systematically decoupled salinity-driven conjugation dynamics through controlled single-factor experiments and simulated sediment microcosms. Controlled conjugation assays revealed a threshold-dependent response, with RP4 plasmid transfer frequencies peaking at 2.00 % salinity (4.58–13.51-fold increase vs. 0.85 % control, p < 0.01), mechanistically linked to reactive oxygen species (ROS)-mediated SOS pathway activation. In simulated sediment systems, salinity gradients drove host-specific ARGs enrichment, with plasmid-borne tetA and blaTEM abundances increasing 1.49–4.39 fold under brackish conditions (2.00 % salinity). Multidrug resistance genes floR, qacH-01 exhibited synergistic diffusion patterns (r = 0.77–0.94, p < 0.05), while salt-tolerant phyla Campylobacterota and Spirochaetota became dominant ARGs reservoirs at 3.50 % salinity (2.05–3.17 fold enrichment vs. controls). Although exogenous antibiotic resistance bacteria (ARB) introduction marginally reduced α-diversity, phylum-level community structure remained stable. Salinity preferentially suppressed rare taxa, amplifying ARGs co-occurrence networks through niche restructuring. These findings establish salinity as a dual regulator of ARGs dissemination, directly enhancing conjugation via oxidative stress pathways and indirectly reshaping resistance landscapes through microbial host selection. The results underscore the necessity of integrating salinity gradients into ARGs risk assessments, particularly in coastal ecosystems where tidal fluctuations may potentiate resistance propagation.