Microbiome and pathogen identification, and associated antimicrobial resistance genes and virulence factors in seafood revealed by 16S rRNA amplicon and metagenomic sequencing

16S rRNA amplicon sequencing and metagenomic next-generation sequencing (mNGS) were employed to comprehensively analyze the microbial communities, foodborne pathogens, antibiotic resistance genes (ARGs), and virulence factors (VFs) in four seafood categories: ready-to-eat fish (RET-fish), non-ready-to-eat fish (non-RTE-fish), shellfish, and shrimp. At the phylum level, Pseudomonadota dominated across all samples. The microbial community composition of shellfish exhibited significant distinctions compared to other seafood categories. Metagenomic profiling identified high-risk pathogens, such as pathogenic Vibrio, Salmonella enterica, and Listeria monocytogenes. ARGs and VFs displayed the highest relative abundance in RET-fish, while shellfish exhibited the lowest abundance with statistically significant differences compared to other groups. For ARGs carried hosts, Bacillus-associated tet(L) and Lactobacillus-associated Inu(A) in RTE-fish demonstrated elevated abundance. In contrast, Vibrio species in other groups carried high abundances of ARGs such as qnrS and tet(34). Additionally, Vibrio harbored high levels of VFs, such as flagella and EF-Tu. Furthermore, plasmid-derived contigs co-harboring ARGs and mobile genetic elements (MGEs) were identified, displaying broad host ranges and high homology with plasmids from previously isolated clinical pathogenic strains, which underscores the potential role of seafood as a critical reservoir for the dissemination of ARGs. High-throughput sequencing approaches, integrated with multi-tool bioinformatics pipelines, provided robust insights into microbial communities and associated safety risk factors. These findings highlight the urgent need for targeted surveillance of seafood products and stricter antibiotic regulations in aquaculture to mitigate public health risks posed by foodborne pathogens and antimicrobial resistance.