Metagenomic and culture-based genomics reveal virulence and resistance risks in Manila clam microbiomes

Bivalves are important aquaculture products whose safety is shaped by their microbiomes. Here, we present the first comprehensive characterization of Manila clam (Ruditapes philippinarum) microbiomes using both shotgun metagenomics (6 clams) and culture-based genomics (169 isolates, 40 draft genomes), integrating community, functional, and antimicrobial resistance profiling. Communities were dominated by Proteobacteria (99.3-99.9 %), with Pseudoalteromonas and Vibrio collectively accounting for 74.9-99.7 % and showing strong inverse correlations, defining Pseudoalteromonas-dominated, Vibrio-dominated, and mixed states. Species richness ranged from 22 to 180 per sample. Recognized human pathogens occurred at low abundance (<0.3 %), including Vibrio parahaemolyticus, Vibrio alginolyticus, and Photobacterium damselae, while opportunistic vibrios expanded in some clams (e.g., Vibrio cyclitrophicus 57.9 %). We reconstructed 34 high-quality MAGs, seven resolved to species (Pseudoalteromonas tetraodonis, V. cyclitrophicus, Shewanella aquimarina), alongside unclassified lineages. Metagenomes encoded 14 virulence-factor categories with 2281 subtypes, and isolate genomes added 93 further subtypes, including high-virulence loci in Escherichia coli and type III secretion genes in V. parahaemolyticus. Resistomes spanned 18 antibiotic classes with 511 subtypes; isolates contributed 22 additional antibiotic resistance genes(ARGs), including extended-spectrum β-lactamases (bla_CTX-M-102) and bla_NDM-1. Four carbapenemase-producing isolates (three Shewanella algae, one V. parahaemolyticus) carried blaNDM-1 on IncC plasmids, with the V. parahaemolyticus plasmid transferable to E. coli. Two P. tetraodonis MAGs encoded RiPP-like and terpene biosynthetic clusters plus phage-defense systems, consistent with Vibrio suppression. These findings demonstrate that clam microbiomes fluctuate between protective (Pseudoalteromonas) and pathogenic (Vibrio-Shewanella) states, providing a first integrated framework for assessing microbial risk, antimicrobial resistance, and food safety interventions in bivalve aquaculture.