Whole genome sequencing of Vibrio harveyi from different sites in the Mediterranean Sea providing data on virulence and antimicrobial resistance genes

Abstract

Vibriosis caused by Vibrio harveyi is an important bacterial infection in the Mediterranean farming of European seabass and gilthead sea bream. Genomic information about V. harveyi contributes to understanding its pathogenesis and virulence. Sixteen V. harveyi strains obtained from diseased seabass and sea bream from Croatia, France, Italy, Spain, Tunisia, and Turkey were sequenced using ONT MinION and Illumina HiSeq. The strains were sequenced, de novo assembled, and the genomes analysed for the presence of plasmids, virulence, and antibiotic resistance genes. Antibiotic resistance genes were compared with in vitro susceptibility testing using disc diffusion and microdilution methods. All studied strains had two circular chromosomes, and twelve strains had one to five circular plasmids. A total of 171 virulence genes were determined using the Virulence Factors of Bacterial Pathogens database (VFBD), 150 virulence genes were present on average in each genome and the distribution of virulence genes was similar across strains. Most of the reported virulence genes have been previously described in bacteria of the genus Vibrio, while genes sitA (4/16), sitB (3/16), sitC (4/16), and sitD (3/16) important for iron transport, and the ast gene (10/16) that encodes a heat-stable cytotoxic enterotoxin, were unique in our studied strains. Using the Comprehensive Antibiotic Resistance database (CARD), it was noted that all strains carried the CRP gene encoding resistance to macrolides, fluoroquinolone, and penams, but showed sensitivity to enrofloxacin and florfenicol using in vitro tests. The adeF gene coding for resistance to fluoroquinolones and tetracyclines was detected in lower percentage in thirteen strains was supported with detected sensitivity to florfenicol and oxytetracycline using in vitro tests. The E.coli parE gene was detected in eleven strains that showed sensitivity to florfenicol and oxytetracycline. The gene APH(3”)Ib responsible for resistance to aminoglycosides was present in one strain, though all strains showed susceptibility to gentamicin by in vitro testing. The Tet(D) gene coding for resistance to tetracyclines was found in a single strain showing reduced sensitivity to oxytetracycline. All tested strains were resistant to ampicillin by in vitro testing in line with previously described innate resistance. Phylogenetic analysis based on whole genome sequences showed a clear separation and clustering of the tested strains from the Mediterranean region. The new genomic data and knowledge provided in this study increase the understanding of the pathogenicity and antimicrobial resistance patterns of V. harveyi strains in the Mediterranean region, enabling a common regional approach to prevention and mitigation of the disease.