The resistome of European coastal environments revealed by high-resolution targeted metagenomics in oysters

Antimicrobial resistance is a major global health threat, and the environment is increasingly recognised as a key reservoir of antibiotic resistance genes (ARGs). In marine ecosystems, filter-feeding organisms such as oysters are sentinel species that reflect the microbiological diversity of their environment. Here, we investigated the ARG content of the Pacific oyster Magallana gigas across four European coastal sites, contrasting anthropogenically impacted aquaculture sites (Bay of Brest, Ebro Delta, Thau lagoon) with natural oyster reefs (Sylt island). ARGs sequencing was enriched using a targeted probe-and-capture metagenomic approach employing 37,826 RNA baits covering over 2000 ARG sequences. Moreover, a novel bioinformatic pipeline ARGifier was specifically designed for sensitive, high-resolution detection and classification of ARGs in metagenomic samples dominated by host DNA. This strategy was able to overcome limitations of cultivation-based methods and whole metagenomic sequencing, enabling the detection of more than 33,000 ARG sequences in the oyster resistome spanning over 450 genes distributed over 23 resistance classes. Reads assigned to ARGs represented 0.23% of all raw reads and 25.02% of host-depleted reads. Accordingly, comparison of targeted metagenomics and pool-seq analysis of cultured isolates (culturomics) in specific pathogen-free oysters revealed that metagenomics captured dominant, community-wide resistance determinants, whereas culturomics recovered a broader diversity of ARGs due to their presence in genomes of cultivable strains. The oyster resistome across the four farming sites was dominated by multidrug efflux systems associated with intrinsic and housekeeping-related mechanisms, suggesting an environmentally shaped resistance profile. Clinically relevant ARGs, including extended-spectrum Beta-Lactamase, carbapenem, colistin, and vancomycin resistance genes, were detected in low abundance at all sites, with higher enrichment in anthropogenically impacted areas. In addition, several ARGs were associated with plasmids, indicating mobilization potential. Taxonomic analysis revealed that a limited subset of bacteria, particularly Betaproteobacteria and Actinomycetes, carried a disproportionately high number of ARGs despite their low relative abundance in the oyster microbiome. Overall, this study delivers a comprehensive inventory of ecologically and clinically relevant ARGs in European coastal environments, positioning farmed and wild oysters as sentinel organisms for monitoring antimicrobial resistance in coastal waters. Our findings highlight the circulation of ARGs within coastal marine ecosystems, which may act as reservoirs and potential sources of mobilisable antimicrobial resistance. Video Abstract