Objective To systematically investigate the genomic structure and functional variations of Pseudomonas plecoglossicida from cultured large yellow croaker, we performed whole-genome resequencing and comparative genomic analysis of clinical isolates and the reference strain ATCC® 700383™, providing a theoretical foundation for developing effective prevention and control strategies.

Methods Whole-genome resequencing was performed on 8 P. plecoglossicida strains isolated from diseased large yellow croaker across different times and locations, along with the reference strain ATCC® 700383™, using the PacBio and Illumina sequencing platforms. Complete circular genomes were assembled and genomic component and functional were annotated. Comparative genomic analysis was conducted using P. plecoglossicida strain BCH2017050402—a representative pathogen of "visceral white spot disease" in large yellow croaker—as the reference, against other P. plecoglossicida strains and closely related species. Additionally, pan-genome analysis has performed on 13 P. plecoglossicida genomes using bioinformatics tools.

Results The complete genome sequences of 8 P. plecoglossicida strains isolated from diseased large yellow croaker ranged from 5,469,808 bp to 5 525 584 bp in size, with GC contents of 62.66%–62.76%, and encoded 4 997–5 045 predicted genes. All strains possessed a single circular chromosome with no plasmids. The representative strain BCH2017050402 had a genome of 5.52 Mb (5,525,533 bp), containing 5,045 genes and 108 non-coding RNAs (ncRNAs). Functional annotation identified 3,814 genes with COG (Clusters of Orthologous Groups) assignments, 4,702 genes mapped to KEGG (Kyoto Encyclopedia of Genes and Genomes) metabolic pathways, 19 genomic islands, and 460 virulence factor genes. The strain also harbored diverse protein secretion systems, including 4 type III secretion system (T3SS) and 10 type VI secretion system (T6SS)-related proteins. The reference strain ATCC® 700383™ had a genome of 5,443,146 bp with 4,954 predicted genes, also organized as a single circular chromosome without plasmids. Comparative genomic analysis revealed 6,727 SNPs between BCH2017050402 and ATCC® 700383™, including 2,012 non-synonymous mutations in coding regions and 51 InDels within CDS (18 insertions, 33 deletions). No large-scale structural rearrangements had be observed, but multiple small-scale structural variations, four large insertions, and several deletions had be detected. High genomic synteny and structural similarity had be observed between BCH2017050402 and other isolates from Ningde (FFH2014050403, FDH2015122403, CZH2019040302, FFH2013032002, and PQLYC4). In contrast, comparison with the Zhejiang isolate XSDHY-P revealed an inverted genomic segment containing one translocation and one translocation-inversion region, along with 12 non-synonymous mutations in coding regions. Synteny was poor when compared with P. putida strain NBRC14164. Pan-genome analysis of 13 P. plecoglossicida genomes showed that the core genome accounted for 88.83% of the pan-genome, with 94 strain-specific genes identified.

Conclusion Genomic sequences of P. plecoglossicida strains isolated from large yellow croaker exhibit extremely high similarity, whereas substantial genomic divergence exists between P. plecoglossicida strains of different origins and their closely related species. Using comparative and pan-genomic approaches, this study characterizes the genomic features of large yellow croaker-derived P. plecoglossicida, elucidating structural variations occurring against a highly conserved core genome background, as well as the distinctive profiles of its virulence and antimicrobial resistance genes.