The genus Vibrio comprises numerous marine bacteria, including several species recognised as important pathogens with significant implications for both human health and aquaculture. In particular, marine molluscs are highly susceptible to infections caused by specific Vibrio taxa. The development of accurate species-level diagnostic tools remains a major challenge, largely due to the substantial intraspecific genetic diversity and the limited availability of complete and curated genome assemblies in public repositories. This taxonomic complexity hinders both pathogen identification and disease management strategies in marine ecosystems.

To address these limitations, we sequenced and assembled 115 complete genomes from strains representing 42 distinct Vibrio species. The genomes were obtained by long-read sequencing and assembled using a dedicated pipeline optimised for long-read data, ensuring high contiguity and completeness. Notably, eight of these genomes represent the first complete assemblies available for their respective species. Various methodologies are currently used for bacterial identification, including sequence-based approaches and MALDI-TOF mass spectrometry.

In this study, we compared the performance of MALDI-TOF, MLSA, and whole genome sequencing for the identification of Vibrio species. Using comparative genomics, we delineated the Vibrio core genome and identified a set of candidate species-specific genomic markers. These loci were systematically validated against a comprehensive dataset of 6,904 publicly available Vibrio genomes from 51 species, with particular emphasis on clades containing mollusc pathogens. Our analysis revealed novel markers with high discriminatory power and interspecies specificity, enabling robust taxonomic resolution across the genus.

We propose a novel single-gene diagnostic marker that allows accurate and consistent identification of Vibrio species, offering a streamlined alternative to current multi-locus approaches. In addition to its diagnostic potential, this marker provides a strong phylogenetic signal, supporting more accurate classification within the genus. This work also allowed us to formulate recommendations for the identification and diagnosis of Vibrio bacteria, contributing to the development of next-generation molecular tools for monitoring Vibrio diversity in marine ecosystems.