Coastal waters are increasingly polluted with antibiotic resistance genes (ARGs) of anthropogenic origin, raising One Health concerns through recreational exposure, aquaculture, and consumption of contaminated seafood, particularly for high-risk ARGs that are human-associated and mobile. At a small coastal watershed (7.5 km²) where the Xichong River discharges directly into Dapeng/Mirs Bay along the Shenzhen-Hong Kong coast, our previous work identified untreated sewage intrusion as the major source of high-risk ARGs, which have also been detected as far as the open South China Sea. However, the specific microbial taxa that acquire, carry, and functionally transmit these genes across the land-ocean continuum remain poorly constrained. To address this, we collected 190 samples between July 2024 and July 2025, spanning river water (n=47), wastewater treatment plant influent and effluent (n=15), estuarine water (n=18), beach groundwater (n=29), and coastal seawater (n=81), to track ARG dynamics across this gradient. We combined culture-independent and culture-dependent approaches to identify the microbial carriers of ARGs and functionally validate their transmission potential. Random forest analysis identified human-associated sewage markers, particularly crAssphage (34.3% relative contribution) and raw sewage bacteria abundance (17.3%), as the top predictors of variation in ARG abundance, establishing raw sewage intrusion as the key factor shaping the watershed resistome. Among 11,093 metagenome-assembled genomes (MAGs) recovered, 1,063 carried ARGs. Several clinically relevant Enterobacteriaceae genera, including Escherichia, Klebsiella, and Enterobacter, harbored multiple ARGs alongside virulence factors, suggesting that these taxa may act as multidrug-resistant opportunistic pathogens in sewage-impacted waters. This enrichment of resistant Enterobacteriaceae was corroborated by MacConkey culture-based screening, which recovered over 130 isolates resistant to last-resort antibiotics (meropenem and ceftazidime). Contig-level co-occurrence patterns and coverage-frequency analyses of 98 high-quality ARG-carrying plasmids further identified Escherichia and Enterobacter as the likely plasmid hosts. Critically, plasmid-mediated conjugation assays showed that native riverine microbial communities could rapidly acquire multidrug-resistance plasmids, at conjugation frequencies ranging from 3.8% to 10.1%. Together, these findings establish Enterobacteriaceae as key reservoirs and active disseminators of ARGs along a sewage-driven pathway connecting land and sea, underscoring the urgency of controlling untreated sewage inputs to curb the spread of clinically relevant antimicrobial resistance in coastal ecosystems.
 

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