Studies on Aeromonas salmonicida subsp. masoucida BR19001YR isolated from diseased black rockfish (Sebastes schlegeli)

Abstract

Aeromonas salmonicida is the cause of furunculosis, causing skin ulcers and eventually mortality in a variety of fish species worldwide. In this study, the major virulence factors were determined by comparing the phenotypic and genotypic characteristics of the pathogenic A. salmonicida BR19001YR isolated from a recent diseased rockfish with the low pathogenic strain A. salmonicida RFAS1. Biochemical property, hemolytic ability, antibiotic susceptibility, and invasive ability were compared to determine the phenotypic factor causing the difference in pathogenicity between the two strains. In order to confirm and compare the exact pathogenicity of BR19001YR and RFAS1, the LD50 was measured. Whole genome sequencing of BR19001YR was performed to confirm the difference in genotypic characteristics by comparative analysis. The difference in pathogenicity was confirmed when the plasmid pBY3 of BR19001YR, which contained the estimated major virulence factors, was transferred to the low pathogenic Edwardsiella piscicida KE2 and the non-pathogenic Edwardsiella tarda SU100 through mating assay. The results show that the BR19001YR is highly pathogenic to rockfish than RFAS1. The biochemical properties and hemolytic ability of BR19001YR were the same as those of RFAS1. In the antibiotic susceptibility test, for tetracycline and sulfonamide in which RFAS1 showed wild type, BR19001YR showed non-wild type and showed 14 times higher invasion ability than RFAS1. RFAS1 did not cause mortality at the concentration of 1.67 * 109 cfu fish-1, whereas the LD50 of BR19001YR was 7.14 * 102 cfu fish-1. As a result of comparative analysis, the p-type type IV secretion system (virB/D4) genes on pBY3, which contains antibiotic resistance determinants, was presumed to be the major virulence factor. When pBY3 was transferred to KE2 through a mating analysis, it was confirmed that the pathogenicity exhibited by the strain to the host was improved. It is strongly inferred that the cause of reduced antibiotic susceptibility and improved invasive ability different from RFAS1 is a novel plasmid pBY3 that has never been reported. It is estimated that BR19001YR obtained pBY3 in seawater environments. This study provided insight into the characterization of highly pathogenic A. salmonicia and virulent plasmids. These results will serve as the basis for an understanding of the mode of action for A. salmonicida, which has not yet been fully elucidated, and will contribute to promoting effective treatment.