Revisiting bioluminescence and sucrose utilization in aquatic pathogens Vibrio harveyi and V. campbellii using genome-wide in silico mapping and phenotyping

Vibrio harveyi is a major bacterial pathogen of shrimp and finfish aquaculture. Traditionally, bioluminescence and sucrose fermentation have served as key phenotypic marker for its identification. However, frequent misidentification with closely related species like V. campbellii necessitates a reassessment of these phenotypic traits. Therefore, these traits were evaluated for genomic distribution, targeted phenotypic validation and its potential role in evolution and speciation. We generated chromosome-level assemblies for seven strains, including V. harveyi SB1 reference genome, and performed genome-wide mapping of 282 strains (204 V. harveyi and 78 V. campbellii), followed by phenotypic validation of 49 isolates. In silico analysis revealed that only 2.9% of V. harveyi strains carry luminescence operon (luxCDABEGH), whereas 100% strains of V. campbellii carried either a functional luxCDABEGH (87.2%) or a defective luxBG operon (12.8%). The functional sucrose operon (scrRAKB) was present in 89.5% strains of V. harveyi (yellow colonies on TCBS agar) but was absent in all V. campbellii (green colony) except strain 170502. Mobilome and synteny analysis revealed horizontal gene transfer of scr operon in 1% strains, while no mobile genetic elements were associated with the luxCDABEGH operon in V. harveyi, despite rare occurrence. Core genome phylogeny indicated that V. harveyi represents an early-evolved lineage, whereas V. campbellii is a recently evolved species within the Harveyi clade. The evolutionary trajectory of V. campbellii further suggests that luminescence-defective strains (e.g., type strain CAIM519T) evolved alongside a group of strains carrying luminescence operon flanked by mobile-genetic elements (e.g., BAA-1116). Phenotypic assays and PCR screening of the luciferase gene (luxA) and sucrose uptake gene (scrA) results were consistent with the genomic findings. Collectively, the present study demonstrates that V. harveyi is predominantly non-luminescent and sucrose-fermenting (yellow), while V. campbellii is primarily luminescent and sucrose non-fermenting (green colonies), providing refined phenotypic criteria for their differential diagnosis.