Tracking a misclassified pathogen: genomic and epidemiological features of Vibrio paracholerae

The genus <i>Vibrio</i> encompasses globally relevant pathogens, of which <i>Vibrio cholerae</i> is the best known due to its role in cholera. Closely related species within the Cholerae clade <i>- Vibrio paracholerae</i>, <i>Vibrio metoecus</i> and <i>Vibrio tarriae</i> - were long misclassified as non-O1/O139 <i>Vibrio cholerae</i>. The objective of this study was to analyse all 13,000+ available <i>V</i>. <i>cholerae</i> genomes in GenBank to determine the presence of species from the Cholerae clade. Genome-wide analyses using Mash, whole-genome-based Average Nucleotide Identity and digital DNA-DNA hybridization reclassified 190 unique genomes as <i>V. paracholerae</i>, while <i>V. metoecus</i> and <i>V. tarriae</i> were not detected. Phylogenomic analyses revealed that <i>V. paracholerae</i> forms distinct lineages, spanning clinical, environmental and animal sources over a period of more than a century. Virulence profiling revealed the absence of cholera toxin and toxin-coregulated pilus; however, most genomes exhibited other virulence factors, including haemolysins, RTX toxins, cholix toxin and a conserved type VI secretion system. Resistome analysis revealed multiple antibiotic resistance genes, several of which were embedded within superintegron regions, reinforcing the role of <i>V. paracholerae</i> as a reservoir of resistance determinants. Importantly, we identified five putative gene markers with high sensitivity and specificity for discriminating the two species, providing a tool for diagnostic applications and epidemiological surveillance. These findings reveal an unsuspected epidemiological scenario for <i>V. paracholerae</i>, which should be considered in clinical monitoring and public health strategies involving the Cholerae clade.