Characterization of the O-antigen gene clusters and development of a molecular serotyping method for Vibrio fluvialis

Vibrio fluvialis is an emerging foodborne pathogen that causes severe infections. Serotyping based on surface polysaccharide antigens is important for the clinical detection and epidemiological surveillance of pathogens such as V. fluvialis. For example, variation of the O-antigen, which is highly polymorphic and is responsible for the majority of antigenic variability on the bacterial cell surface, provides the basis for serotyping of Gram-negative bacteria. Currently, there has been no analysis of the O-antigen gene clusters in V. fluvialis. In this study, the putative O-antigen gene clusters of 18 V. fluvialis serogroups (O1-O18), which exhibit a high level diversity, were analyzed by whole-genome sequencing. A microsphere-based suspension array (MSA) based on O-serogroup-specific genes was developed for identification of V. fluvialis strains O1-O18 and evaluated for specificity and sensitivity in double-blind tests. Furthermore, analysis of 62 publicly available V. fluvialis genomes identified 13 new O-antigen gene cluster types. The detection sensitivity was determined to be 10^-2 ng for genomic DNA and 10³ CFU for pure cultures. When testing simulated samples in an oyster background, 2 to 20 CFU per gram inoculated could be detected after enrichment using this method. Our work provides an efficient tool for rapid detection and identification of V. fluvialis serogroups from clinical and environmental samples, with the potential for use in epidemiological investigations and food safety applications.