Non-Pandemic Vibrio cholerae and Vibrio parahaemolyticus ST98 Recovered from Commercially Depurated Bivalves Marketed in Portugal: Whole-Genome and Phylogenomic Characterization

Vibrio cholerae and Vibrio parahaemolyticus are naturally occurring aquatic bacteria of public-health relevance that may contaminate seafood, including bivalve mollusks. This study characterized two Vibrio isolates recovered from commercially depurated mussels (Mytilus spp.) and clams (Spisula solida) marketed at retail in Portugal. Species identification, antimicrobial susceptibility testing, Oxford Nanopore whole-genome sequencing (WGS), average nucleotide identity analysis, antimicrobial resistance and virulence profiling, genoserotyping, prophage prediction, and core-genome phylogenomic analysis were performed. WGS confirmed the mussel-derived isolate as V. cholerae and the clam-derived isolate as V. parahaemolyticus. Phenotypic testing demonstrated meropenem resistance in the V. cholerae isolate, whereas several additional antimicrobial MICs could not be categorically interpreted because of panel concentration limits or the absence of Vibrio-specific clinical breakpoints. The V. cholerae isolate lacked ctxA, ctxB, the toxin-coregulated pilus cluster, ace, and zot, while its O1/O139 serogroup status remained unresolved because an O1-associated marker was absent and a complete but non-exact wbfZ-like sequence was detected. Its genome nevertheless contained an extensive Type VI secretion system repertoire together with several hemolysin- and toxin-associated determinants. Phylogenomically, the isolate was most closely related to the environmental strain RFB16 and was distinct from the pandemic reference lineages included in the analysis. The V. parahaemolyticus isolate was assigned to ST98 and OL4 and carried a divergent KL55-like K-antigen locus. Its virulence-associated repertoire was dominated by a well-conserved Type III secretion system 1, together with adhesion-, motility-, and hemolysin-associated determinants, whereas the major enteropathogenic markers tdh and trh were absent. It grouped most closely with strain vp-HL-202008, although this relationship received moderate phylogenetic support. Resistance-associated determinants were predominantly chromosomal, and prophage analysis identified two putative regions in V. cholerae and seven in V. parahaemolyticus. Overall, these findings characterize two Vibrio isolates recovered from commercially depurated bivalves marketed in Portugal. Their detection does not demonstrate depuration failure or an immediate consumer-health risk but highlights that conventional indicator-based compliance does not necessarily exclude autochthonous Vibrio spp. and supports the value of genomic surveillance in seafood safety.