Vibrio alginolyticus is a frequently implicated species for vibriosis in humans and diverse wildlife, but it has previously been difficult to identify from the closely related and emerging Vibrio diabolicus. Comparisons of both species, including antimicrobial resistance (AMR) and virulence characterizations, are scarce and impeded by intraspecies diversity, minimal genomes, discordant classification methods, and gene databases with limited utility to understudied species. The species identities of 3,442 public domain genomes (SRA files) within the Harveyi clade were re-evaluated using genomic methods. Public genomes identified as V. diabolicus and V. alginolyticus were combined with previously published genomes isolated from humans, sea otters (Enydra lutris), or coastal environments (V. diabolicus n = 88, V. alginolyticus n = 163, Vibrio parahaemolyticus n = 287) for pangenome-wide association studies to identify species-specific gene clusters (95% identification threshold). Additional genome wide associations with isolation source (humans versus sea otters) were investigated, including AMR and virulence related gene clusters. Genomic reclassification identified 29 of 150 misclassified public domain V. alginolyticus genomes, including 26 reclassified as V. diabolicus. In total, 28 previously misclassified V. diabolicus genomes (n = 37 total) were identified, including 10 human-derived strains. GWAS identified 643 and 477 gene clusters specific to V. alginolyticus and V. diabolicus, respectively, while some multilocus sequencing analysis (MLSA) gene clusters were non-specific. Gene clusters (n = 109) associated with either V. alginolyticus isolated from humans or sea otters were identified including one annotated to a multidrug resistance gene (mdtk_1). No V. diabolicus gene clusters were associated with host species after multiple comparison correction, although pre-correction associations related to antimicrobial resistance were detected (cat_1, ampC). The genomic methods of classification presented provide accurate species identification for V. diabolicus and V. alginolyticus beyond current MLSA/MLST schemes, although target species-specific genes were identified that may be useful for improved future schemes. While limited sample size of V. diabolicus hampered the ability to detect host associated markers, the GWAS approach employed provide a reusable framework for discovering insights into host adaptation and prioritizing target genes for future functional AMR and virulence validation experiments in both species.
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